U.S. patent application number 13/239662 was filed with the patent office on 2012-05-03 for antimicrobial compounds and formulations.
This patent application is currently assigned to LYTIX BIOPHARMA AS. Invention is credited to Bengt Erik Haug, Istvan Marko, Oystein Rekdal, Merete Linchausen Skar, Wenche Stensen, Morten Bohmer Strom, John Sigurd Svendsen.
Application Number | 20120108520 13/239662 |
Document ID | / |
Family ID | 9887297 |
Filed Date | 2012-05-03 |
United States Patent
Application |
20120108520 |
Kind Code |
A1 |
Svendsen; John Sigurd ; et
al. |
May 3, 2012 |
Antimicrobial Compounds and Formulations
Abstract
The invention relates to the use of a molecule comprising a
backbone of 2 to 35 non-hydrogen atoms in length, having covalently
attached thereto at least two bulky and lipophilic groups and
having at least one more cationic than anionic moiety, in the
manufacture of a medicament for destabilising microbial cell
membranes and the use as a membrane acting antimicrobial agent of a
molecule comprising a backbone of 2 to 35 non-hydrogen atoms in
length, having covalently attached thereto a super bulky and
lipophilic group comprising at least 9 non-hydrogen atoms and
having at least two more cationic than anionic moieties and to
methods of treatment involving such molecules, in particular
peptides including peptide derivatives, and peptidomimetics.
Inventors: |
Svendsen; John Sigurd;
(Kvaloysletta, NO) ; Haug; Bengt Erik; (Tromso,
NO) ; Marko; Istvan; (B-Louvain-la-Neuve, BE)
; Rekdal; Oystein; (Tomasjord, NO) ; Skar; Merete
Linchausen; (Tromso, NO) ; Stensen; Wenche;
(Kvaloysletta, NO) ; Strom; Morten Bohmer;
(Tromso, NO) |
Assignee: |
LYTIX BIOPHARMA AS
Tromse
NO
|
Family ID: |
9887297 |
Appl. No.: |
13/239662 |
Filed: |
September 22, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11738098 |
Apr 20, 2007 |
8048852 |
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13239662 |
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10221040 |
Feb 27, 2003 |
7232803 |
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PCT/GB01/01035 |
Mar 9, 2001 |
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11738098 |
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Current U.S.
Class: |
514/19.2 |
Current CPC
Class: |
A61P 31/04 20180101;
A61K 38/08 20130101; A61P 35/00 20180101; C07K 5/0821 20130101;
A61K 38/07 20130101; Y02A 50/30 20180101; C07K 5/06095 20130101;
A61K 47/54 20170801; C07K 5/1019 20130101; A61K 38/06 20130101;
C07K 5/0817 20130101; C07K 5/1024 20130101; A61K 47/543 20170801;
C07K 5/06026 20130101; C07K 5/06086 20130101; A61P 31/12 20180101;
A61P 31/10 20180101; A61K 38/05 20130101; A61K 47/64 20170801; C07K
5/06139 20130101; C07K 7/06 20130101 |
Class at
Publication: |
514/19.2 |
International
Class: |
A61K 38/03 20060101
A61K038/03; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 9, 2000 |
GB |
0005703.4 |
Claims
1-40. (canceled)
41. A method of treating tumors comprising administration of an
effective amount of a molecule of 2 to 4 amino acids or equivalent
subunits in length, which incorporates a bulky and lipophilic group
comprising at least 13 non-hydrogen atoms and containing no more
than 2 polar functional groups, wherein said bulky and lipophilic
group incorporates one or more closed rings of 5 or more
non-hydrogen atoms, said molecule further having at least two more
cationic than anionic moieties to a human or animal patient.
42. A method as claimed in claim 41, wherein said bulky group
incorporates 2 closed rings of 5 or more non-hydrogen atoms.
43. A method as claimed in claim 41 wherein said molecule is a
peptide, peptide derivative or peptidomimetic.
44. A method as claimed in claim 43, wherein said molecule
comprises a modified C terminus which does not carry a negative
charge.
45. A method as claimed in claim 44, wherein said C terminus is
amidated or esterified.
46. A method as claimed in claim 44, wherein said modified C
terminus comprises a bulky and lipophilic group.
47. A method as claimed in claim 45, wherein said modified C
terminus comprises a bulky and lipophilic group.
48. A method as claimed in claim 43 wherein said peptide, peptide
derivative or peptidomimetic comprises one or more amino acid or
equivalent R subunits which comprises a bulky and lipophilic
group.
49. A method as claimed in claim 48, wherein one or more of said
amino acid or subunits comprises a cationic moiety.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 11/738,098, filed Apr. 20, 2007 which is a continuation of Ser.
No. 10/221,040, filed Feb. 27, 2003, now U.S. Pat. No. 7,232,803,
issued Jun. 19, 2007, which is a 371 filing of PCT/GB/01035, filed
Mar. 9, 2001, which claims priority from GB 0005703.4, filed Mar.
9, 2000. All of these prior applications are incorporated herein by
reference.
[0002] The present invention relates to bioactive molecules, in
particular to small molecules which exhibit antimicrobial
activity.
[0003] Peptides and their derivatives have long been recognised as
therapeutically interesting molecules. A wide variety of organisms
use peptides as part of their host defense mechanism. Antimicrobial
peptides have been isolated from species as diverse as bacteria and
mammals [Lehrer, R. I., Lichtenstein, A. K. and Ganz, T. (1993)
Ann. Rev. Immunol. 11, 105-128]. Generally, these peptides have a
net positive charge and a propensity to form amphiphilic
.alpha.-helix or .beta.-sheet structures upon interaction with the
outer phospholipid bilayer in bacterial cell membranes [Besalle,
R., Gorea, A., Shalit, J., Metger, J. W., Dass, C. Desiderio, D. M.
and Fridkin, M. (1993) J. Med. Chem. 36 1203-1209]. In most cases
the detailed molecular mechanisms of the antibiotic action are
unknown, although some peptides categorised as class L (lytic)
peptides are believed to interact with bacterial cell membranes,
probably forming ion-channels or pores [Ludtke, S. J., He, K.,
Heller, W. T., Harroun, T. A., Yang, L. and Huang, H. W. (1996)
Biochemistry 35 13723-13728] leading to permeability changes and
consequent cell lysis.
[0004] Magainins are antibacterial peptides from the skin of the
frog Xenopus laevis and are classified as class L antibiotics
because they specifically lyse bacteria; other peptides such as
mastroparans, a bee venom, lack this specificity as they lyse
eukaryotic as well as prokaryotic cells and are called Class L
Venoms [Tytler, E. M., Anantharamaiah, G. M., Walker, D. E.,
Mishra, V. K., Palgunachari, M. N. and Segrest, J. P. (1995)
Biochemistry 34 4393-4401].
[0005] As well as magainins and mastroparans, host defense peptides
have been isolated from moths and flies (cecropins) and from
Horseshoe crab. The direct action of these host defense peptides to
repel predators, for example as venoms, is clear. The search for
peptides which exhibit antibiotic effects has lead to the
identification of other proteins/peptides which would not be
expected to have cytotoxic properties. One of these is lactoferrin,
an iron transporter which also shows a weak antibacterial
effect.
[0006] The majority of known antibacterial peptides comprise 10 or
more, typically 20 or more amino acids, this number of amino acid
being required in order to provide sufficient length for the
peptide, generally in .alpha.-helical form, to span the bacterial
cell membrane and form a pore. Such a mechanism is the generally
accepted way in which the majority of such peptides exert their
cytotoxic activity.
[0007] Synthesis of the antibacterial peptides of the prior art can
be difficult, and typically requires the peptides to be synthesised
by bacteria or other organisms which can be cultured and harvested
to yield the peptide of interest, additional processing steps after
isolation of the direct product of translation are generally
required. If active peptides could be identified which were
shorter, this would enable economic manufacture by synthesis from
the amino acid building blocks or available di- or tri-peptides. In
addition, short peptides would offer advantages for biodelivery.
There is a growing demand for antibiotics which can be administered
without the need for an injection, such as by inhalation and
absorption across the blood capillaries of the nasal passages.
Thus, an object of the present invention is to provide bioactive,
particularly antimicrobial e.g. antibacterial, molecules which are
small enough to be synthesised without the need to transfect
organisms with nucleic acid encoding for the peptide of interest
and which offer a variety of different modes of administration.
[0008] The search for novel antibiotics has taken on particular
urgency because of the increasing number of bacterial strains which
are exhibiting resistance to known and extensively used drugs.
Those operating in the fields of medicine as well as agriculture,
environmental protection and food safety are constantly requiring
new antibacterial agents and may have to treat a given population
or site with several different antibacterial agents in order to
effectively combat the undesirable bacteria.
[0009] All peptides, and this applies even more so to short
peptides, are susceptible to enzymatic degradation in the human or
animal body. Therefore, peptide derivatives or peptidomimetics
which retain or even enhance the biological activity of the basic
peptide but have a greater circulating half life would be
particularly advantageous and the provision of such compounds
constitutes a further object of the present invention.
Peptidomimetics and other organic molecules may be readily
synthesised in large amounts by non-fermentation methods.
[0010] Combinatorial libraries have been used to identify active
peptides (Blondelle, S. E. et al. [1994] American Society for
Microbiology Vol. 38, No. 10, 2280-2286). While a vast number of
peptides can be screened in this way, the reasons behind the
activity of one peptide compared to another may not be clear. An
anomalous result indicating activity for a particular sequence may
encourage research into a class of molecules which as a whole do
not represent the best therapeutic candidates. In addition, with
combinatorial chemistry it is often difficult to be sure about
exactly what compounds have actually been made and laborious
testing and analysis is required to confirm identity of
manufactured compounds. If one is looking to identify a core active
motif which may not be sequence or size dependent, combinatorial
techniques are unsuitable. Also, the chemistry used in building up
the molecule, typically from monomers must be rather simple,
limiting the variety of molecules which can be made.
[0011] In the present case, the inventors have sought to
investigate what structural components are required in order to
provide the desired therapeutic and general antimicrobial activity,
while limiting toxicity and enabling relatively straightforward
manufacture and flexibility in terms of the routes of
administration of the active molecules. The techniques used, rather
than an undirected production and analysis of thousands, even
millions of molecules, akin to looking for a needle in a haystack,
have been based on rational design. The inventors have sought to
identify important motifs and those components which are both
necessary and sufficient to the provision of molecules with the
desirable characteristics discussed above. Such an approach has
proved effective and is particularly valuable in enabling
identification of the smallest, simplest molecules possible which
can be synthesised and are preferably resistant to enzymatic
degradation, i.e. are not underivatised peptides.
[0012] It has surprisingly been found that small molecules,
equivalent to 4 amino acids or less, exhibit good bioactivity
provided they possess sufficient bulky and lipophilic and cationic
groups. Previously, it had been thought that only larger molecules,
typically longer peptides, could exhibit the desired therapeutic
activity, as a result of the way such molecules were believed to
exert their effect on cell membranes. It is particularly surprising
that these small molecules exhibit good selectivity, i.e. they are
cytotoxic against microbes but have very little, if any, toxic
activity against host eurkaryotic cells.
[0013] Thus, according to one aspect of the present invention is
provided a bioactive molecule comprising a backbone of 2 to 35,
typically 4 to 35, preferably 4 to 20, more preferably 4 to 12,
e.g. 6 to 9 non-hydrogen atoms in length, having covalently
attached thereto at least two bulky and lipophilic groups and
having at least one more cationic than anionic moiety for use in
therapy, e.g. as an antimicrobial, particularly as an antibacterial
agent.
[0014] This definition could encompass short unmodified peptides
but such peptides which only contain amino acids selected from the
20 genetically coded amino acids and also have no bulky or
lipophilic N or C terminal modifications are not included within
the scope of this aspect of the present invention. The purpose of
the present invention is not to identify active peptide fragments
per se but to provide stable active molecules which can be prepared
by chemical synthesis.
[0015] Such antimicrobial molecules also have non-therapeutic uses,
for example in agriculture or in domestic or industrial situations
as sterilising agents for materials susceptible to microbial
contamination. Thus, in a further aspect, the present invention
provides the use of a bioactive molecule comprising a backbone of 2
to 35, typically 4 to 35, preferably 4 to 20, more preferably 4 to
12, e.g. 6 to 9 non-hydrogen atoms in length, having covalently
attached thereto at least two bulky and lipophilic groups and
having at least one more cationic than anionic moiety as an
antimicrobial, particularly as an antibacterial agent.
[0016] The molecules exhibit antimicrobial activity, in particular
they exert a cytotoxic effect through a direct membrane-affecting
mechanism and can be termed membrane acting antimicrobial agents.
These molecules are lytic, destabilising or even perforating the
cell membrane. This offers a distinct therapeutic advantage over
agents which act on or intereact with proteinaceous components of
the target cells, e.g. cell surface receptors. While mutations may
result in new forms of the target proteins leading to antibiotic
resistance, it is much less likely that radical changes to the
lipid membranes could occur to prevent the cytotoxic effect. The
lytic effect causes very rapid cell death and thus has the
advantage of killing bacteria before they have a chance to
multiply. In addition, the molecules may have other useful
properties which kill or harm the target microbes e.g. an ability
to inhibit protein synthesis, thus they may have multi-target
activity.
[0017] Thus, the invention also provides the use of a bioactive
molecule comprising a backbone of 2 to 35, typically 4 to 35,
preferably 4 to 20, more preferably 4 to 12, e.g. 6 to 9
non-hydrogen atoms in length, having covalently attached thereto at
least two bulky and lipophilic groups and having at least one more
cationic than anionic moiety in the manufacture of a medicament
having a membrane acting antimicrobial activity. This mode of
action means, that while the molecules of the invention may be
administered in conjunction with other active antimicrobial agents
as part of a combined therapy, they may also be administered on
their own, i.e. as the sole antimicrobial agent in a therapeutic
regimen. This can be contrasted, for example, with molecules acting
as efflux pump inhibitors which are co-administered with a primary
antimicrobial agent, often having no antimicrobial activity of
their own.
[0018] Thus in a preferred embodiment of the invention is provided
the use of the molecules defined herein in the manufacture of a
medicament for destabilising and/or permeabilising microbial cell
membranes. In other words the molecules are provided for use in the
destabilisation of microbial cell membranes. By `destabilisation`
is meant a perturbation of the normal three dimensional lipid
bi-layer configuration including but not limited to membrane
thinning, increased membrane permeability (typically not involving
channels) of water, ions or metabolites etc. which also impairs the
respiratory systems of the bacteria. The mechanisms for bacterial
lysis caused by antimicrobial peptides are extensively reviewed by
Sitaram and Nagaraj (N. Sitaram and R. Nagaraj, Biochim. Biphys.
Acta vol 1462 1999 p. 29-54) and Shai (Y. Shai, Biochim. Biophys.
Acta vol 1462 1999 p. 55-70). Destabilisation kills or weakens the
cell making it less likely to grow or reproduce.
[0019] As discussed above, the inventors in this case have sought
to identify those functional and structural motifs which together
give the molecules the desired properties of therapeutic
(antimicrobial) activity but low toxicity. In a preferred
embodiment of the present invention, a third type of group is also
found in the molecules, the first two being positively charged
groups and bulky and lipophilic groups. This third group is a
carbonyl or similar polar group such as a sulphone, thio carbonyl
or imine. Such a group is a hydrogen bond acceptor moiety and may
conveniently be found as part of the backbone of the molecule, for
example the amide bonds found in peptide or other backbones, other
backbones may comprise ester or thioester linkages which give the
desired polarity to the molecule.
[0020] The `length` of the backbone is the shortest distance in
terms of number of atoms between the two atoms in the backbone
which are furthest apart. The two atoms which are furthest apart
are those which are separated from each other by the greatest
number of covalent bonds. Thus, if to get from one of the two atoms
which are furthest apart to the other it is necessary to pass
through 6 further atoms, the backbone is 8 atoms in length.
Hydrogen atoms are not considered to be atoms of the backbone which
will typically comprise carbon, nitrogen or oxygen, possibly
sulphur or phosphorous atoms. The backbone may be linear, branched,
cyclic or polycyclic.
[0021] The backbone may contain one or more cyclic groups but the
bulky and lipophilic groups defined herein are not considered to
form part of the backbone. The backbone is generally characterised
by forming a non-interrupted chain of atoms, including chains
forming a closed ring or rings, to which the bulky and lipophilic
and cationic groups are attached. By `non-interrupted` it is meant
that the backbone is continuous, with the bulky and lipophilic
groups attached thereto rather than interrupting the chain of
backbone atoms. Preferably, the atoms of the backbone will form a
linear or branched chain.
[0022] Thus the following molecule would have a backbone of 8 atoms
in length, following the method for calculating backbone length
defined above.
##STR00001##
[0023] The following molecule would also have a backbone of 8
atoms; as discussed below atoms forming cationic moieties may be
part of the backbone.
##STR00002##
[0024] The backbone will typically only comprise less than 4 atoms
when one or more of the bulky and lipophilic groups is attached
directly to the backbone so that a single atom is part of a defined
bulky and lipophilic group as well as the backbone. When an atom is
shared in this way, this atom is functionally part of the bulky and
lipophilic group and is not counted as an atom of the backbone.
Such a molecule is shown below; this molecule thus has a 2 atom
backbone.
##STR00003##
[0025] By `bulky and lipophilic` group is meant an uncharged group
of at least 4, preferably at least 5, more preferably at least 6
non-hydrogen atoms, typically incorporating at least one closed
ring system. For convenience, such groups are sometimes referred to
herein simply as `bulky` groups. One or more of the bulky and
lipophilic groups present in the molecule may have 2 or more closed
rings of 5 or 6 atoms and conveniently 2 or more of these rings are
fused or bridged. Preferably, at least one of the bulky and
lipophilic groups is not provided by the unmodified R group of one
of the 20 genetically coded amino acids. Aromatic bulky and
lipophilic groups are preferred, as are groups which are three
dimensional in character. If the group does not contain one or more
rings then it will preferably be branched.
[0026] It appears that the positioning of the functional groups
(e.g. charged or bulky groups) is not of great importance. The
bulky and lipophilic groups have a combined impact on the activity
of the molecule as a whole. Thus a comparatively small group
together with a rather large group may contribute a similar
activity to 2 moderately sized bulky groups. Thus while an example
of the minimum bulk present in the molecule is 2 tert.-butyl
groups, if one such or similarly sized group is present, a second
larger bulky group will preferably be incorporated.
[0027] A preferred lower limit of bulk for the molecules defined
herein is therefore a tert.-butyl group or equivalent
(trimethylsilyl for example is only slightly larger) and a
6-membered ring, e.g. a cyclohexyl or phenyl group. A hierarchy of
bulky groups can be exemplified by the following list of amino
acid, starting with the least bulky and active: tert.-butylglycine,
phenylalanine, cyclohexylalanine, tryptophan,
tert.-butylphenylalanine, biphenylalanine and the most bulky and
active, tri tert.-butyltryptophan. The skilled reader will
appreciate that such groups are given as examples of different
sizes and other groups of a similar volume may be used as a
substitute without significantly affecting the molecule's
activity.
[0028] Preferably the molecule will incorporate two groups the size
of a phenyl group or larger, i.e. 6-membered rings or equivalent
(e.g. --CH.sub.2C(CH.sub.3).sub.3). Particularly preferably, one
bulky group is a phenyl group (or equivalent) or larger i.e. has 6
or more non-hydrogen atoms and the other has 9 or more non-hydrogen
atoms, e.g. as provided by the R group of tryptophan,
tert.-butylphenylalanine or biphenylalanine having 10, 11 and 13
non-hydrogen atoms respectively. It should be remembered that the
necessary number and nature of the molecule's bulky groups will
vary from one type of microorganism to another, with a given
molecule generally much more active against Gram-positive than
Gram-negative bacteria.
[0029] By a `cationic moiety` is meant a moiety which has a net
positive charge at pH 7.0 or a precursor of such a moiety which is
capable of providing in physiological conditions a moiety which has
a net positive charge at pH 7.0. Such precursor moieties being
known in the art. Likewise an `anionic moiety` is one which has a
net negative charge at pH 7.0 or a precursor thereof. Positive
charges are important for attraction to and interaction with the
negatively charged phospholipids which make up cell membranes.
Suitable chemical groups which provide this cationic functionality
include those which comprise an ammonium, guanidino, imidazolium,
sulphonium or phosphonium moiety or a tetrazole.
[0030] A cationic moiety may be incorporated as part of a bulky and
lipophilic group, e.g. a modified tryptophan residue such as
5'-aminoethyltryptophan (available as side chain Boc and N-alpha
FMOC derivative from RSP Amino Acids Analogues Inc, Boston, Mass.,
USA). The atom which actually carries the positive charge when the
molecule is at pH 7.0 may be spaced from the backbone. For example,
consider the R group of arginine, here the whole R group is
considered to be the cationic moiety and the atoms attaching the
guanidino group to the a carbon atom are thus considered to be part
of the cationic moiety and not part of the backbone.
[0031] By way of example, the molecule Arg-Trp OBz, a dipeptide
whose C terminus has been modified by formation of a benzoyl ester
has one cationic moiety supplied by the R group of arginine and one
at the free N terminus. The anionic C terminus has been modified,
thus the molecule has two more cationic moieties than anionic
moieties, i.e. 2 additional cationic moieties. Likewise, if the N
terminus had been modified, for example, by a cyclohexylcarboxylate
group, then a cationic moiety would have been `lost`.
[0032] A group which is responsible for increasing the cationicity
of the molecule through modification of the C terminus may also
provide one of the bulky and lipophilic groups, as in the above
example.
[0033] A nitrogen atom, for example one which forms part of a
cationic ammonium group at the N terminus of a peptide may be one
of the backbone atoms. Thus the cationic moieties may form part of
the backbone or be appended thereto.
[0034] The molecules for use according to the invention will
typically have one or more, preferably 2 or more cationic moieties
but it is important to consider the number of both cationic and
anionic moieties present. For example the tri-peptide Trp-Arg-Trp
has two cationic moieties, one supplied by the R group of arginine
and the N terminal group. However the anionic C terminus is not
modified so the molecule as a whole has only one more cationic
moiety than anionic moiety.
[0035] Throughout the text, the well known 3 letter and 1 letter
codes for the genetically coded amino acids are used.
[0036] Preferably, the bioactive molecules of the invention will
comprise two or more bulky and lipophilic groups and two or more
additional cationic moieties (additional being used to indicate the
number of extra cationic moieties present in the molecule as
compared to anionic moieties). The inventors have identified the
presence of two additional cationic moieties and two bulky and
lipophilic groups as one motif which provides particularly active
molecules, although further bulky and/or cationic groups may also
be present.
[0037] Alternatively, molecules incorporating at least three bulky
and lipophilic groups and at least one additional cationic moiety,
e.g. three bulky and lipophilic groups and one additional cationic
moiety, have also been shown to possess good activity and this is a
further particularly preferred motif. Cationicity or bulk alone do
not provide the desired activity. Similarly, three additional
cationic moieties in a molecule with just one bulky and lipophilic
group does not provide the desired level of bioactivity, unless the
bulky and lipophilic residue is `super` bulky and lipophilic.
[0038] Without wishing to be bound by theory, it seems that the
`super bulky and lipophilic group` is exerting the same influence
on the molecule as two regular bulky and lipophilic group. By
`super bulky and lipophilic group` is meant a group of at least 9,
typically at least 10 or 11, preferably at least 12 or 13, more
preferably at least 15 or 18 non-hydrogen atoms which comprises 1
or more, preferably 2 or more closed ring systems of 4 or more
non-hydrogen atoms each, e.g. the R group of tri-tert.butyl
tryptophan, di-tert-butyl tryptophan or PMC
(2,2,5,7,8-pentamethylchroman-6-sulphonyl) modified tryptophan or
adamantylalanine. The super bulky group preferably comprises at
least the equivalent of one 6 membered ring attached to a
tert.-butyl group e.g. a tert.-butylphenyl group. More preferred
are groups comprising two fused or more particularly non-fused 5 or
6 membered rings, e.g. naphtyl, diphenylmethyl, biphenyl or larger
groups.
[0039] Thus, in a further aspect, the present invention provides a
bioactive molecule comprising a backbone of 2 to 35, typically 4 to
35, preferably 4 to 20, more preferably 4 to 12, e.g. 6 to 9
non-hydrogen atoms in length, having covalently attached thereto at
least one super bulky and lipophilic group and comprising at least
two more cationic than anionic moieties for use in therapy, e.g. as
an antimicrobial, as an particularly antibacterial agent. As
before, these molecules are membrane acting antimicrobial
agents.
[0040] Further aspects of the invention include the use as
non-therapeutic agents of these molecules; suitable non-therapeutic
uses which utilise the general antimicrobial activity of these
molecules are discussed herein.
[0041] These molecules may also comprise one or more regular bulky
and lipophilic groups as described above, covalently attached to
the backbone.
[0042] Preferred amongst the bioactive molecules described above
are peptides which incorporate 1-4 amino acids, preferably 2 or 3
amino acids but also conveniently 4 amino acids. The amino acids
may be genetically coded amino acids, genetically coded amino acids
which have been modified or modified or non-modified
non-genetically coded amino acids which may or may not be naturally
occurring. .beta. and .gamma. amino acids as well as a amino acids
are included within the term `amino acids`. Peptides may be cyclic
in nature. The term `peptide` includes depsi peptides.
[0043] Typically these peptide or peptide derived molecules will
incorporate N- and/or C-terminal modifying groups. The bulky and
lipophilic groups may be provided by the R groups of the amino acid
residues and/or be part of the N- or C-terminal modifying group.
The cationic moieties may be free N-terminal groups, amino acid R
groups or part of the N or C terminal modifying groups. The
C-terminus is preferably modified, e.g. amidated or more preferably
esterified. The use of the term amino acid `R group` is well
understood in the art and used consistently throughout the text to
refer to the variable group attached to the .alpha.-carbon atom,
e.g. for alanine a methyl group.
[0044] Thus, a preferred type of backbone for the bioactive
molecules described herein will be peptidic or peptide like.
Peptidic backbones are characterised by the
##STR00004##
linkage, a peptide or amide bond. Peptide backbones incorporate at
least one such peptide bond. Backbones which terminate in a peptide
bond, e.g. an amidated carboxy group are not considered peptidic
purely on the basis of this group. Thus, to be classed as peptidic,
the backbone must have one or more internal peptide bonds.
[0045] While a peptidic backbone is characterised by one or more
internal peptide bonds, a peptide will have peptide bonds linking
each amino acid residue.
[0046] Thus, a compound wherein one or more amide bond has been
replaced by an alternative linker but wherein at least one amide
bond remains will have a peptidic backbone as defined herein but
the compound as a whole will not be a peptide but a
peptidomimetic.
[0047] KIM/Peptide-like (peptidomimetic) backbones are a further
class of suitable backbones and may be preferred, for example
because they can offer the molecule as a whole resistance to
hydrolytic enzymes. Peptidomimetic backbones will generally be
linear or linear strings of fused cyclic groups which mimic the
peptide backbone.
[0048] A peptidomimetic is typically characterised by retaining the
polarity, three dimensional size and functionality (bioactivity) of
its peptide equivalent but wherein the peptide bonds have been
replaced, often by more stable linkages. By `stable` is meant more
resistant to enzymatic degradation by hydrolytic enzymes.
Generally, the bond which replaces the amide bond (amide bond
surrogate) conserves many of the properties of the amide bond, e.g.
conformation, steric bulk, electrostatic character, possibility for
hydrogen bonding etc. Chapter 14 of "Drug Design and Development",
Krogsgaard, Larsen, Liljefors and Madsen (Eds) 1996, Horwood Acad.
Pub provides a general discussion of prior art techniques for the
design and synthesis of peptidomimetics. In the present case, where
the molecule is reacting with a membrane rather than the specific
active site of an enzyme, some of the problems described of exactly
mimicking affinity and efficacy or substrate function are not
relevant and a peptidomimetic can be readily prepared based on a
given peptide structure or a motif of required functional groups.
Suitable amide bond surrogates include the following groups:
N-alkylation (Schmidt, R. et al., Int. J. Peptide Protein Res.,
1995, 46,47), retro-inverse amide (Chorev, M. and Goodman, M., Acc.
Chem. Res, 1993, 26, 266), thioamide (Sherman D. B. and Spatola, A.
F. J. Am. Chem. Soc., 1990, 112, 433), thioester, phosphonate,
ketomethylene (Hoffman, R. V. and Kim, H. O. J. Org. Chem., 1995,
60, 5107), hydroxymethylene, fluorovinyl (Allmendinger, T. et al.,
Tetrahydron Lett., 1990, 31, 7297), vinyl, methyleneamino (Sasaki,
Y and Abe, J. Chem. Pharm. Bull. 1997 45, 13), methylenethio
(Spatola, A. F., Methods Neurosci, 1993, 13, 19), alkane (Lavielle,
S. et. al., Int. J. Peptide Protein Res., 1993, 42, 270) and
sulfonamido (Luisi, G. et al. Tetrahedron Lett. 1993, 34,
2391).
[0049] The peptidomimetic compounds of the present invention may
have one or more, preferably 2 or 3 identifiable sub-units which
are approximately equivalent in size and function to amino acids.
The term `amino acid` may thus conveniently be used herein to refer
to the equivalent sub-units of a peptidomimetic compound. Moreover,
peptidomimetics may have groups equivalent to the R groups of amino
acids and discussion herein of suitable R groups, including
modified R groups and of N and C terminal modifying groups applies,
mutatis mutandis, to peptidomimetic compounds.
[0050] As is discussed in the text book referenced above, as well
as replacement of amide bonds, peptidomimetics may involve the
replacement of larger structural moieties with di- or
tripeptidomimetic structures and in this case, mimetic moieties
involving the peptide bond, such as azole-derived mimetics may be
used as dipeptide replacements. Peptidomimetics and thus
peptidomimetic backbones wherein the amide bonds have been replaced
as discussed above are, however, preferred.
[0051] Suitable peptidomimetics include reduced peptides where the
amide bond has been reduced to a methylene amine by treatment with
a reducing agent e.g. borane or a hydride reagent such as lithium
aluminium-hydride. Such a reduction has the added advantage of
increasing the overall cationicity of the molecule.
[0052] Other peptidomimetics include peptoids formed, for example,
by the stepwise synthesis of amide-functionalised polyglycines.
Some peptidomimetic backbones will be readily available from their
peptide precursors, such as peptides which have been permethylated,
suitable methods are described by Ostresh, J. M. et al. in Proc.
Natl. Acad. Sci. USA (1994) 91, 11138-11142. Strongly basic
conditions will favour N-methylation over O-methylation and result
in methylation of some or all of the nitrogen atoms in the peptide
bonds and the N-terminal nitrogen.
[0053] Preferred peptidomimetic backbones include polyesters,
polyamines and derivatives thereof as well as substituted alkanes
and alkenes. The peptidomimetics will preferably have N and C
terminii which may be modified as discussed herein.
[0054] Peptides and peptidomimetics will generally have a backbone
of 4 to 20, preferably 7 to 16 atoms in length. Molecules having
backbones at the upper end of these ranges will generally comprise
.beta. and/or .gamma. amino acids or their equivalents.
[0055] Typically, the peptides for use as antimicrobial agents
according to the invention will include 2 or 3 amino acids, at
least one of which has a cationic R group. Suitable genetically
coded amino acids which provide this cationic functionality would
therefore be lysine, arginine and histidine, non-genetically coded
amino acids and modified amino acids which also provide a cationic
R group include analogues of lysine, arginine and histidine such as
homolysine, ornithine, diaminobutyric acid, diaminopimelic acid,
diaminopropionic acid and homoarginine as well as trimethylysine
and trimethylornithine.
[0056] One or more of the amino acid residues may have an R group
which provides one of the required bulky and lipophilic groups. Of
the genetically coded amino acids, tryptophan, phenylalanine and
tyrosine are particularly suitable and leucine, isoleucine and
methionine may also be used. Tryptophan, because of its two fused
ring structure and additional bulk is particularly preferred,
although the polarity of tyrosine may also be useful. Non-genetic
amino acids, which may be naturally occurring, and tryptophan,
phenylalanine and tyrosine analogues and amino acids which have
been modified to incorporate a bulky and lipophilic R group may
also be used. All such modified and unmodified amino acids may
conveniently be referred to as `bulky and lipophilic amino
acids`.
[0057] The closed ring systems are typically formed of carbon
atoms, optionally also including nitrogen, oxygen or sulphur atoms.
Particularly preferred amino acids comprise a substituted or
unsubstituted indole. The R group may preferably be
three-dimensional. Preferred amino acids incorporating a bulky and
lipophilic R group include adamantylalanine, 3-benzothienylalanine,
4,4'-biphenylalanine, 3,3-diphenylalanine, homophenylalanine,
2,6-dichlorobenzyltyrosine, cyclohexyltyrosine,
7-benzyloxytryptophan, tri-tert.-butyltryptophan, homotryptophan,
3-(-anthracenyl)-L-alanine, L-p-iso-propylphenylalanine,
L-thyroxine, 3,3'5-triiodo-L-thyronine, triiodo-tyrosine.
[0058] A lipophilic molecule is one which associates with its own
kind in an aqueous solution, not necessarily because the
interactions between the lipophilic molecules are stronger than
between the lipophilic molecule and water but because interactions
between a lipophilic molecule and water would destroy the much
stronger interactions between the water molecules themselves. It is
therefore preferable that the bulky and lipophilic R group should
not contain many polar functional groups e.g. no more than 4,
preferably 2 or less. Such groups would increase the binding
interaction with the aqueous surroundings and hence lower the
lipophilicity of the molecule. For example, a phenyl group as a
component of a bulky and lipophilic group may be preferred to a
pyridyl group, even though they have the same number of
non-hydrogen atoms and are of a similar overall size. However, the
presence of a hydroxyl group in a bulky and lipophilic group has
been shown to enhance activity and particularly in longer peptide
and peptidomimetic compounds, one or more of the bulky and
lipophilic groups will preferably contain one or two polar groups,
particularly hydroxy groups. Thus amphipathic groups such as
phenolic groups may be particularly effective bulky and lipophilic
groups, especially in longer molecules.
[0059] Non-genetic bulky and lipophilic amino acids include
modified tryptophan, tyrosine and phenylalanine residues, in
particular tryptophan residues which have been substituted at the
1-, 2-, 5- and/or 7-position of the indole ring, positions 1- or
2-being preferred e.g. 5' hydroxy tryptophan. A variety of other
amino acid derivatives having a bulky and lipophilic character are
known to the man skilled in the art.
[0060] Suitable amino acids include thyroxine and the following
commercially available amino acids and their derivatives:
[0061] L-3-benzothienylalanine, CAS=72120-71-9 (Synthetech),
D-3-benzothienylalanine, CAS=111139-55-0 (Synthetech),
L-4,4'-biphenylalanine (Synthetech), D-4,4'-biphenylalanine
(Synthetech), L-4-bromophenylalanine, CAS=24250-84-8 (Synthetech),
D-4-bromophenylalanine, CAS=62561-74-4 (Synthetech),
L-2-chlorophenylalanine, CAS=103616-89-3 (Synthetech),
D-2-chlorophenylalanine, CAS=80126-50-7 (Synthetech),
L-3-chlorophenylalanine, CAS=80126-51-8 (Synthetech),
D-3-chlorophenylalanine, CAS=80126-52-9 (Synthetech),
L-4-chlorophenylalanine, CAS=14173-39-8 (Synthetech),
D-4-chlorophenylalanine, CAS=14091-08-8 (Synthetech),
L-3-cyanophenylalanine, CAS=57213-48-6 (Synthetech),
D-3-cyanophenylalanine (Synthetech), L-4-cyanophenylalanine
(Synthetech), D-4-cyanophenylalanine (Synthetech),
L-3,4-dichlorophenylalanine, CAS=52794-99-7 (Synthetech),
D-3,4-dichlorophenylalanine, CAS=52794-98-6 (Synthetech),
L-3,3-diphenylalanine (Synthetech), D-3,3-diphenylalanine
(Synthetech), L-homophenylalanine, CAS=943-73-7 (Synthetech),
D-homophenylalanine, CAS=82795-51-5 (Synthetech),
L-2-indanylglycine (Synthetech), D-2-indanylglycine (Synthetech),
L-4-iodophenylalanine, CAS=24250-85-9 (Synthetech),
D-4-iodophenylalanine, CAS=62561-75-5 (Synthetech),
L-1-naphthylalanine, CAS=55516-54-6 (Synthetech),
D-1-naphthylalanine, CAS=78306-92-0 (Synthetech),
L-2-Naphthylalanine, CAS=58438-03-2 (Synthetech),
D-2-naphthylalanine, CAS=76985-09-6 (Synthetech),
L-3-trifluoromethylphenylalanine, CAS=14464-68-7 (Synthetech),
D-3-trifluoromethylphenyl-alanine (Synthetech),
L-4-trifluoromethylphenylalanine, CAS=114926-38-4 (Synthetech),
D-4-trifluoromethyl-phenylalanine, CAS=114872-99-0 (Synthetech),
Boc-D-homophenylalanine (Neosystem Laboratoire),
Boc-L-homophenylalanine (Neosystem Laboratoire),
Fmoc-4-methyl-D-phenylalanine (Neosystem Laboratoire),
Fmoc-4-methyl-L-phenylalanine (Neosystem Laboratoire),
2,6-dichlorobenzyltyrosine, CAS=40298-71-3 (Senn Chemicals),
Benzyltyrosine Fmoc (Senn Chemicals), Cyclohexyltyrosine Fmoc (Senn
Chemicals), L-3,5-diiodotyrosine, CAS=300-39-0 (Senn Chemicals),
D-3,5-diiodotyrosine (Senn Chemicals), L-3,5-dibromotyrosine (Senn
Chemicals), D-3,5-dibromotyrosine (Senn Chemicals),
L-t-butyltyrosine (Senn Chemicals), L-t-butyltyrosine (Senn
Chemicals), N-Acetylhomotryptophan (Toronto Research),
7-Benzyloxytryptophan (Toronto Research), Homotryptophan (Toronto
Research), 3-(-Anthracenyl)-L-alanine Boc (or Fmoc) (Peninsula
Laboratories), 3-(3,5-Dibromo-4-chlorophenyl)-L-alanine (Peninsula
Laboratories), 3-(3,5-Dibromo-4-chlorophenyl)-D-alanine (Peninsula
Laboratories), 3-(2-Quinoyl)-L-alanine Boc (or Fmoc) (Peninsula
Laboratories), 3-(2-Quinoyl)-D-alanine Boc (or Fmoc) (Peninsula
Laboratories), 2-Indanyl-L-glycine Boc (Peninsula Laboratories),
2-Indanyl-D-glycine Boc (Peninsula Laboratories),
L-p-t-butoxyphenylglycine Fmoc (RSP), L-2-t-butoxyphenylalanine
Fmoc (RSP), L-3-t-butoxyphenylalanine Fmoc (RSP), L-homotyrosine,
O-t-butyl ether Fmoc (RSP), L-p-t-butoxymethylphenylalanine Fmoc
(RSP), L-p-methylphenylalanine Fmoc (RSP), L-p-ethylphenylalanine
Fmoc (RSP), L-p-iso-propylphenylalanine Fmoc (RSP),
L-p-methoxyphenylalanine Fmoc (RSP), L-p(tBu-thio)phenylalanine
Fmoc (RSP), L-p-(Trt-thiomethyl)phenylalanine Fmoc (RSP),
L-p-hydroxymethyl-phenylalanine, O-t-butyl (RSP),
L-p-benzoylphenylalanine (Advanced ChemTech),
D-p-benzoylphenylalanine (Advanced ChemTech), O-benzyl-L-homoserine
Boc (Advanced ChemTech), O-benzyl-D-homoserine Boc (Advanced
ChemTech), L-.beta.-1-Naphthyl-alanine (Advanced ChemTech),
D-.beta.-1-Naphthyl-alanine (Advanced ChemTech),
L-penta-fluorophenylalanine Boc (Advanced ChemTech),
D-penta-fluorophenylalanine Boc (Advanced ChemTech),
D-penta-fluorophenylalanine Fmoc (Advanced ChemTech),
3,5-Diiodo-L-tyrosine Fmoc (Boc) (Advanced ChemTech), L-Thyroxine
Na, CAS=6106-07-6 (Novabiochem), 3,3', 5-Triiodo-L-thyronine Na,
CAS=55-06-1 (Novabiochem).
[0062] Surprisingly, it has been found that standard chemical
protecting groups when attached to an amino acid R group can
provide suitable bulky and lipophilic groups. Such modified R
groups constitute preferred bulky and lipophilic groups. Suitable
amino acid protecting groups are well known in the art and include
Pmc (2,2,5,7,8-pentamethylchroman-6-sulphonyl), Mtr
(4-methoxy-2,3,6-trimethylbenzenesulfonyl) and Pbf
(2,2,4,6,7-pentamethyldihydrobenzofuransulfonyl), which may
conveniently increase the bulk and lipophilicity of aromatic amino
acids, e.g. Phe, Trp and Tyr. Also, the tert.-butyl group is a
common protecting group for a wide range of amino acids and is
capable of providing bulky and lipophilic groups as described
herein, particularly when modifying aromatic residues. The Z-group
(carboxybenzyl) is a further protecting group which can be used to
provide a bulky and lipophilic group.
[0063] A bulky and lipophilic group as defined above may also be
provided by an N terminal modifying group. Such bulky and
lipophilic N-terminal modifications will preferably comprise a 5-
or 6-membered ring which may be alkyl or aryl e.g.
cyclohexylcarboxylate or benzylcarboxylate. The bulky and
lipophilic N-terminal modifying group may encompass 2 or more fused
rings one or more of which may be a 5-membered ring e.g. adamantyl
or indole. In addition, due to its tendency to cause unacceptable
levels of toxicity (i.e. haemolytic activity) and to provide
peptides which are bacteriostatic rather than bactericidal, Fmoc is
excluded from possible bulky and lipophilic N terminal
modifications. N terminal acetyl groups are not preferred for
similar reasons.
[0064] Suitable molecules which could be used to modify the
N-terminus and provide a bulky and lipophilic group include:
[0065] cis-Bicyclo[3.3.0]octan-2-carboxylic acid, [18209-43-3]
(Aldrich); Abietic acid, [514-10-3] (Aldrich); Ursolic acid,
[77-52-1] (Aldrich); (1,2-Methanofullerene C.sub.60)-61-carboxylic
acid, [155116-19-1] (Fluka); Dimethyl cubane-1,4-dicarboxylate,
[29412-62-2] (Fluka); 2-Norbornaneacetic acid, [1007-01-8]
(Aldrich); 4-Pentylbicyclo[2.2.2]octane-1-carboxylic acid,
[73152-70-2] (Aldrich); Adamantyl acetic acid;
3-Noradamantanecarboxylic acid, [16200-53-6] (Aldrich);
9-Fluoreneacetic acid, [6284-80-6] (Aldrich);
cis-Decahydro-1-naphthol, [36159-47-4] (Aldrich);
9-Ethyl-bicyclo[3.3.1]nonane-9-ol, [21915-33-3] (Aldrich);
3-Quinuclidinol, [1619-34-7] (Aldrich); [[(1S)-endo]-(-)-Borneol,
[464-45-9] (Aldrich); (1R,2R,3R,5S)-(-)-Isopinocampheol,
[25465-65-0] (Aldrich); Dehydroabietylamine [1446-61-3] (Aldrich);
(.+-.)-3-Aminoquinuclidine [6530-09-2] (Aldrich);
(R)-(+)-Bornylamine, [32511-34-5] (Aldrich);
1,3,3-Trimethyl-6-aza-bicylo[3.2.1]octane [53460-46-1] (Aldrich);
1-Adamantylamine, [768-94-5] (Aldrich); 9-Aminofluorene,
[5978-75-6] (Aldrich); (1R)-(-)-10-Camphorsulfonic acid,
[35963-20-3] (Aldrich); 5-Isoquinolinesulfonic acid, [27655-40-9]
(Aldrich); 2-Quinolinethiol, [2637-37-8] (Aldrich);
8-Mercaptomenthone, [38462-22-5] (Aldrich).
[0066] N-terminal modifications which provide bulky and lipophilic
groups will therefore typically comprise a bulky and lipophilic
group "R" which may be attached directly to the N-terminal amine to
form a mono-, di- and possibly cationic trialkylated N-terminal
amine. Alternatively, the R group may be attached via a linking
moiety e.g. a carbonyl group (RCO) e.g. adamantyl or benzyl,
carbamate (ROCO), or a linker which forms urea (RNHCO) or
(R.sub.2NCO) or by a linker which forms a sulfonamide, boronamide
or phosphonamide. Sulfonamide forming linkers may be particularly
useful when a more stable peptide is required. The bulky and
lipophilic group R comprises a preferably saturated cyclic group,
more preferably a polycyclic group wherein the cyclic groups are
fused or bridged.
[0067] A bulky and lipophilic group as defined above may also be
provided by a C-terminal modifying group. Suitable C-terminal
modifications include the formation of esters, including thioesters
or substituted primary and secondary amides to form e.g. a benzyl
or cyclohexyl ester or amide. In general, esters are preferred.
Other bulky and lipophilic C-terminal groups include naphthylamine
and substituted aromatic amines such as phenyl-ethylamine. Standard
C-terminal protecting groups may also provide a bulky and
lipophilic group.
[0068] C-terminal modifications will therefore typically comprise a
bulky and lipophilic group "R" which may be attached directly to
the C-terminal carboxy group to form a ketone. Alternatively, the R
group may be attached via a linking moiety, e.g. (OR) which forms
an ester at the C-terminus, (NH--R) or (NR.sub.2, wherein the two R
groups needs not be the same) which form primary and secondary
amide groups respectively at the C-terminus or groups
(B--(OR).sub.2) which form boronic esters or phosphorous analogs.
Dae (diaminoethyl) is a further linking moiety which may be used to
attach a bulky and lipophilic group, e.g. carbobenzoxy (Z) to the
C-terminus.
[0069] C-terminal modifications have the advantage of `removing` an
anionic group and thus increasing the cationic nature of the
molecule as a whole. Therefore, while the cationic N-terminus will
generally not be modified unless by a bulky and lipophilic group,
the C-terminus will typically be modified either by the
incorporation of a bulky and lipophilic group or otherwise to
negate the negative charge, e.g. by amidation or formation of a
non-bulky and lipophilic ester e.g. an alkyl ester such as a methyl
ester. In this way, the peptide Tbt-Arg-Trp-NH.sub.2 can have the
desirable 2 bulky and lipophilic groups (provided by Tbt and Trp)
and 2 cationic groups, at the N-terminus and the R group of
arginine, neither of which are `negated` by an anionic
C-terminus.
[0070] A moderately bulky C terminal group, such as a group
comprising a single, preferably 6-membered, ring such as a group
forming a benzyl ester has been shown to provide peptides with
particularly good therapeutic properties and peptides comprising
such group thus make up a preferred group of molecules according to
the present invention.
[0071] Thus, according to a further aspect, the present invention
provides artificial peptides (peptide derivatives) of 1 to 4 amino
acids, typically 2, 3 or 4 amino acids in length which incorporate
at least 2 bulky and lipophilic groups (or at least one super bulky
and lipophilic group) and have at least one more cationic than
anionic moiety. Preferably the peptides incorporate at least 2
bulky and lipophilic groups (or at least 1 super bulky and
lipophilic group) and at least two more cationic than anionic
moieties or at least 3 bulky and lipophilic groups and at least one
more cationic than anionic moiety. The molecules for use according
to the invention are preferably peptides, including peptide
derivatives or peptidomimetics and they are preferably
non-cyclic.
[0072] Peptidomimetic equivalents of the above peptides constitute
a further aspect of the present invention. Such a peptidomimetic
molecule may contain one or more internal amide bonds and the
backbone of such a molecule, would as discussed above thus be
considered peptidic although as a result of other amide bonds or
other modifications, the molecule is not `a peptide`.
[0073] The terms `bulky and lipophilic`, `super bulky and
lipophilic` as well as the definitions of cationic and anionic
groups are as described previously. These short peptides will
preferably be modified at the N and/or C terminus. The peptides are
referred to as `artificial peptides` to indicate that peptides
incorporating only amino acids selected from the 20 genetically
coded amino acids and no bulky and lipophilic N or C terminal
modification are not intended to be covered within the scope of
this aspect of the invention. In addition, as discussed above due
to its tendency to cause unacceptable levels of toxicity (i.e.
haemolytic activity) Fmoc is excluded from possible bulky and
lipophilic N terminal modifications.
[0074] There will be practical upper limits on how bulky and
lipophilic a group can be particularly in terms of increasing
toxicity of the molecule to unacceptable levels. This may be
dependent on the overall size of the molecule and factors such as
three dimensionality of the group and the total number of
non-hydrogen atoms in the group as well as its position within the
molecule as a whole, i.e. whether it is a terminal or internal
group.
[0075] The present invention, as well as providing a group of
compounds for use in therapy and novel bioactive molecules per se,
also provides a method of drug identification and production based
on the functional motifs identified herein. It has surprisingly
been found that very small molecules, such as small peptides can
have excellent therapeutic, e.g. antimicrobial activity but that
such activity is dependent on the presence of a certain number of
bulky and lipophilic and cationic moieties; suitable motifs for
these functional groups are defined herein. Identification of these
motifs provides a very useful strategy for those seeking to prepare
antimicrobial molecules and particularly allows the preparation of
molecules which are smaller than conventional therapeutic
antimicrobial agents. Potential lead candidate drug compounds may
be identified, and optionally further modified to enhance
activity.
[0076] Thus, in a further aspect, the present invention provides a
process for the preparation of a membrane acting antimicrobial
agent comprising identifying a peptide of 1 to 4 amino acids in
length having at least one more cationic than anionic moieties and
having at least two bulky and lipophilic groups or groups which
could be modified to provide bulky and lipophilic groups and
synthesising a derivative or a peptidomimetic of said peptide which
has a backbone of 2 to 35, typically 4 to 35, preferably 4 to 20,
more preferably 4 to 12, e.g. 6 to 9 non-hydrogen atoms in length,
having covalently attached thereto at least two bulky and
lipophilic groups and having at least one more cationic than
anionic moiety and optionally formulating said peptide, peptide
derivative or peptidomimetic with a physiologically acceptable
carrier or excipient.
[0077] The initially identified molecule may be a peptide such as a
fragment of a known peptide or a fragment synthesised de novo. This
peptide may be tested for its biological activity and then the
synthesizing step performed before testing of the peptide,
derivative or peptidomimetic of the invention. Preferably, the
initially identified peptide will not be synthesised and tested but
will simply provide the basis for synthesis of a molecule according
to the present invention. That molecule may itself be tested and
then further modified in accordance with the teaching herein. Prior
to synthesis, there will be a design process where the precise
nature and position of the functional groups and the necessary
synthetic steps are determined.
[0078] More generally, the present invention provides a process for
the preparation of an antimicrobial or antitumoural agent which
method comprises identifying a compound comprising a backbone of 2
to 35, typically 4 to 35, preferably 4 to 20, more preferably 4 to
12, e.g. 6 to 9 non-hydrogen atoms in length, having covalently
attached thereto at least two bulky and lipophilic groups and
having at least one more cationic than anionic moiety and
synthesising said compound and optionally formulating said compound
with a physiologically acceptable carrier or excipient.
[0079] This method also applies to those molecules which comprise
only one super bulky and lipophilic group.
[0080] It has also been observed that the incorporation of one or
more enantiomeric amino acids can significantly increase the
bioactivity of the peptides, such peptides would also have reduced
susceptibility to enzymatic hydrolysis. Thus one or more of the
amino acids present in the molecule may be in the D-form, e.g. all
amino acids may be in the D form, alternate residues may be in the
D form or there may be blocks of D and L residues.
[0081] Suitable compounds which have the structural and functional
characteristics of the bioactive molecules of the present invention
but which are not peptides or peptidomimetics may be readily
prepared by the man skilled in the art. In this case, the
`backbone` typically provides a scaffold onto which the cationic
and bulky and lipophilic groups i.e. the functional groups
responsible for the molecule's activity are attached.
Peptidomimetic molecules are described above and may provide useful
therapeutic compounds but the present invention also relates to
molecules which are not closely based on a standard peptide
structure.
[0082] The `backbone` may be simply a linker moiety which joins the
different functional groups together and provides the required
spacing to allow the cationic and bulky/lipophilic moieties to
perform their roles of attraction to and destabilisation of the
cell membrane. Depending on the particular bulky and lipophilic and
cationic moieties selected, a certain amount of backbone structure
will be required to give the molecule chemical stability, such
considerations being very familiar to the man skilled in the art.
The backbones of such molecules may be linear, branched, cyclic or
polycyclic, aromatic or aliphatic, possibly based on a sugar or
sugar derived compound such as a sugar alcohol or amino sugar,
aminoglycoside, glycoside, aza sugar, innositol, mannitol,
sphingoside or polyester or polyamine.
[0083] The backbones will typically comprise carbon, nitrogen,
oxygen, sulphur or phosphorous atoms but may be further
substituted. Preferably, the backbones will be stable and rather
unreactive under normal physiological conditions, resistant to
enzymatic cleavage and having few charged or polar groups. The
backbone will preferably be biocompatible. These non-peptide like
backbones (i.e. not peptide or peptidomimetic) will have a backbone
length of 2-35 non-hydrogen atoms and where the backbones are
polycyclic e.g. cyclodextrins may actually contain a great many
more non-hydrogen atoms. Preferred backbones will be 4 to 24 e.g. 7
to 16 non-hydrogen atoms in length.
[0084] From a synthetic point of view, the majority of suitable
non-peptide like backbones may conveniently be divided into two
classes, a scaffold type backbone, typically a simple molecule
which has a sufficient number of appendage points for incorporation
of the necessary cationic and bulky and lipophilic moieties. Linear
and cyclic sugars, polyols and inositols fall within this category
and may be exemplified by mannitol which has had its hydroxy groups
modified by the addition of bulky and lipophilic and cationic
moieties. Such molecules may also be formed by the reaction of two
or more distinct components, e.g. the formation of an ester by the
reaction of arginine and mandelic acid. The basic structure or
backbone scaffold may be formed in this way and the molecule
optionally modified to incorporate further cationic and bulky and
lipophilic groups. These scaffold backbones will preferably be
cyclic, e.g. a 4-20 membered ring more typically comprising 6-20
e.g. 9-12 non-hydrogen atoms.
[0085] The purpose of the scaffold molecule is to present the
functional groups e.g. cationic or bulky and lipophilic groups, in
a position necessary for bioactivity. The scaffold molecule must
therefore be able to constrain the topology of the moieties
responsible for the bioactivity. One such suitable scaffold
molecule is a highly functionalised small (5-7 membered) ring of
defined steretochemistry [Luthman, K. and Hacksell, U., A Textbook
of Drug Design and Development, Krogsgaard-Larsen, Liljefors and
Madsen (Eds.) Harwood Academic Press (1996) 9, 386]. In order to
prepare the final molecule a suitable protected scaffold molecule
must be chosen. The synthesis will then typically proceed as
follows: first one of the preferred moieties is linked to the
scaffold typically by ester, ether, amide or amine bond, the next
appendage point in the scaffold molecule is deprotected and
connected to the next preferred moiety as described above. The
process of deprotection and connection is repeated until the
required number of functional groups is obtained. The techniques of
protection and deprotection are well known to the man skilled in
the art and can also be found in the literature [Greene, T. W. and
Wuts, P. G. M., Protective Groups in Organic Synthesis, 2nd ed.,
John Wiley & Sons, Inc. 1991].
[0086] An example of a scaffold molecule and its functionalised
analog is shown below.
##STR00005##
[0087] As well as sugar based scaffold backbones, macrocyclic
amines such as tri- and tetraaza macrocyclic amines (e.g.
1,4,7-triazacyclononane and 1,4,7,10-tetraazacyclodecane) are also
particularly suitable and are readily derivatised at the N atoms to
incorporate the necessary functional moieties as discussed
above.
[0088] Alternatively, the molecule may be built up from similar
monomer sub-units, although such compounds will often be classed as
peptidomimetics as discussed above.
[0089] Molecules may be constructed using a `jigsaw` technique of
`interlocking` i.e. reactive subunits which typically each comprise
a portion which will form the backbone of the molecule as well as
carrying a functional group, i.e. a cationic or bulky and
lipophilic moiety. The produced bioactive molecule may be linear
comprising a chain of monomer subunits or provide a cyclic or
polycyclic structure, which may be 3-dimensional. This particularly
provides a convenient alternative to decorating a basic scaffold
backbone in the synthesis of more complex molecules which do not
comprise repeating similar monomer subunits. Such techniques are
known in the art.
[0090] Suitable bulky and lipophilic groups and cationic moieties
are discussed above and a large number of specific examples are
given in relation to N and C terminal modifying and amino acid R
groups. The same and similar bulky and lipophilic and cationic
moieties may be incorporated in the non-peptide like molecules. For
non-peptide like molecules particularly suitable bulky and
lipophilic groups include.
[0091] The bioactive molecules for use according to the invention
will preferably combine good activity against target pathogens e.g.
as measured by MBC values and comparatively low toxicity as
measured by hemolytic activity. Thus the molecules will preferably
have an MBC against S. aureus of 50 .mu.g/ml or less, more
preferably 20 .mu.g/ml or less and a hemolytic activity of
EC.sub.50 .gtoreq.500 .mu.g/ml, preferably .gtoreq.1000
.mu.g/ml.
[0092] The principles which led to identification of the above
described molecules have been used to identify slightly larger
bioactive molecules, based on peptides of 5 or 6 amino acids in
length. Here the motif of bulky and lipophilic and cationic
moieties identified which provides good activity is at least 2
bulky and lipophilic groups, preferably 3 such groups and at least
2 cationic moieties, preferably 3 or 4 such moieties. Suitable
bulky and lipophilic and cationic moieties are as defined above in
relation to the smaller molecules.
[0093] These peptides are further characterised in that at least
one of the bulky and lipophilic or cationic moieties is not
provided by a genetically coded bulky and lipophilic or cationic
amino acid such as tryptophan, phenylalanine, tyrosine, arginine,
lysine or histidine. Thus, this moiety, which is conveniently
referred to herein as an `artificial bulky and lipophilic moiety`
or `artificial cationic moiety` may be provided by the R group of a
non-genetically coded bulky and lipophilic amino acid such as
tri-tert.-butyl tryptophan or by the R group of a non-genetically
coded cationic amino acid such as homoarginine. Suitable
non-genetically coded amino acids may be naturally occurring or
synthetic and are exemplified herein in relation to the smaller
molecules. An artificial bulky and lipophilic moiety may also
conveniently be provided by modification of the R group of a
genetically coded or non-genetically coded amino acid, e.g. with
PMC or another protecting group. The modified amino acid may itself
be a bulky and lipophilic amino acid such as tryptophan. Again,
suitable modified residues are discussed above in relation to the
smaller molecules.
[0094] Alternatively or in addition an artificial bulky and
lipophilic moiety may be provided by an N or C terminal modifying
group such as have already been described herein. The peptides may
incorporate a bulky and lipophilic moiety at both the N and C
terminii, at either the N or C terminus or at neither terminus.
Where only one terminus carries a bulky and lipophilic moiety, that
will preferably be the C terminus. If the C terminus is not
modified by incorporation of a bulky and lipophilic group as
defined herein it will preferably be otherwise modified to remove
the negative charge normally present at the C terminus at pH 7.0.
Suitable C terminal modifications will include amidation or
formation of an ester which does not include a bulky and lipophilic
moiety, e.g. a short chain alkyl ester such as a methyl ester.
Preferably, at least one of the bulky and lipophilic moieties is an
artificial bulky and lipophilic moiety.
[0095] The artificial bulky and lipophilic moiety will preferably
be at least as bulky and lipophilic, if not more bulky and
lipophilic, than the bulky and lipophilic R group of any
genetically coded amino acid, i.e. at least as bulky and lipophilic
as tryptophan. The enhanced bulkiness and lipophillicity resulting
in peptides which are highly antimicrobially active. The activity
of these peptides would appear to be sequence independent, the
presence of particular functional groups (cationic and bulky and
lipophilic) are responsible for the molecules' cytotoxic
activity.
[0096] These peptides are preferably synthesised by standard
methods of peptide synthesis from the individual amino acid
building blocks. Modified residues may be incorporated during
synthesis but the residues may alternatively be modified after
synthesis of the full peptide. Non-genetically coded or modified
amino acids, aside from any residue incorporating an `artificial
bulky and lipophilic moiety` or an `artificial cationic moiety`,
may be incorporated but preferably the peptide will include some or
a majority of genetically coded residues. Post synthetic
modification may be used to provide an artificial bulky and
lipophilic moiety.
[0097] Thus, in a further aspect, the present invention provides
bioactive peptides of 5 or 6 amino acids in length which
incorporate at least 2 bulky and lipophilic moieties and at least 2
cationic moieties, wherein at least one of said bulky and
lipophilic moieties is an artificial bulky and lipophilic moiety or
at least one of said cationic moieties is an artificial cationic
moiety. The use of these peptides as antimicrobial or antitumoural
agents and pharmaceutical and other compositions containing them
constitute further aspects of the present invention. Of the
genetically coded amino acids, arginine, lysine and histidine are
cationic residues and tyrosine, phenylalanine, tryptophan, leucine,
isoleucine and methionine are bulky and lipophilic residues. 6
residues are preferred and if only 5 amino acids are present,
preferably 3 of these are bulky and lipophilic in character.
[0098] Peptidomimetic compounds having the structural and
functional characteristics of the peptides described above may be
prepared and constitute, together with their uses as antimicrobial
and antitumoural agents further aspects of the present
invention.
[0099] These 5 and 6 mer peptides and compositions, particularly
pharmaceutical compositions comprising them for use in therapy,
e.g. as antitumoural or antimicrobial, particularly antibacterial
agents constitute further aspects of the present invention. As
discussed previously, there are a range of non-therapeutic uses of
active antimicrobial agents and these uses constitute further
aspects of the present invention.
[0100] In a yet further aspect of the present invention, a class of
small peptides incorporating all genetically coded amino acids have
been identified with good bioactivity. Thus, the present invention
provides bioactive peptides of 5 or 6 amino acids in length which
have an unmodified N terminus, all of said amino acids being either
cationic or bulky and lipophilic in nature, at least two amino
acids being bulky and lipophilic and at least two being
cationic.
[0101] Peptides in this category are described in Examples 1 and 4.
It should be recognised that arginine is used as an example of a
genetically coded cationic amino acid and tryptophan or tyrosine as
an example of a genetically coded bulky and lipophilic amino acid.
The other genetically coded bulky and lipophilic and cationic amino
acids have been described previously. Equivalents of the peptides
of Examples 1 and 4 incorporating other genetically coded bulky and
lipophilic amino acids in place of tryptophan and/or arginine are
included within this aspect of the invention. The C terminus of
these peptides is unmodified or amidated or esterified with a small
non-bulky and lipophilic group. Pharmaceutical compositions
comprising these peptides and their use as antimicrobial or
antitumoural agents constitute further aspects of the present
invention.
[0102] The molecules of the invention typically have an
antimicrobial e.g. antibacterial, antiviral or antifungal activity.
In addition, the molecules exhibit antitumoural activity, the
molecules selectively lysing cancer cells rather than healthy
eukaryotic cells. The molecules may be lytic, and/or cause a
destabilisation of the cell membrane which can effect permeability
and cell viability. The molecules are active against Gram negative
and Gram positive bacteria but have been shown to be particularly
effective against Gram-positive bacteria. Thus the uses, therapies
and medicaments are preferably for the treatment of a Gram-positive
infection.
[0103] The molecules may be bactericidal or bacteriostatic,
bactericidal molecules generally being preferred. A high MBC value
but a low MIC value is indicative of a bacteriostatic molecule; the
dipeptide TbtR OMe for example is bacteriostatic in respect of E.
coli. The tripeptide RTbtR OMe which incorporates an additional
cationic group is bactericidal. Increasing the cationicity of a
molecule is a tool which may be used to provide a bactericidal
molecule and thus, in a further aspect, the present invention
comprises a method of increasing the bactericidal activity of a
peptide as compared to its bacteriostatic activity, said peptide
having 2-4 amino acids, at least one more cationic than anionic
moiety and at least one super bulky and lipophilic group or at
least two bulky and lipophilic groups by increasing by at least one
the number of cationic moieties present in the peptide. In general
it seems that the presence of at least two, e.g. 3 or 4 additional
cationic groups provides active molecules but cationicity can be
reduced if the number of bulky and lipophilic groups is increased
to compensate.
[0104] The invention therefore provides methods of treating
microbial infections by administering the various molecules
described herein. In particular methods of destabilising microbial
cell membranes are provided. The amount administered should be
effective to kill all or a proportion of the target microbes or to
prevent or reduce their rate of reproduction or otherwise to lessen
their harmful effect on the body. The clinician or patient should
observe improvement in one or more of the parameters or symptoms
associated with the infection. Administration may also be
prophylactic.
[0105] The peptides of the invention may be synthesised in any
convenient way. Generally the reactive groups present (for example
amino, thiol and/or carboxyl) will be protected during overall
synthesis. The final step in the synthesis will thus be the
deprotection of a protected derivative of the invention. As
discussed above, certain peptides of the invention will carry a
`protecting group` as this is responsible for enhanced
cytotoxicity.
[0106] In building up the peptide, one can in principle start
either at the C-terminal or the N-terminal although the C-terminal
starting procedure is preferred.
[0107] Methods of peptide synthesis are well known in the art but
for the present invention it may be particularly convenient to
carry out the synthesis on a solid phase support, such supports
being well known in the art.
[0108] A wide choice of protecting groups for amino acids are known
and suitable amine protecting groups may include carbobenzoxy (also
designated Z) t-butoxycarbonyl (also designated Boc),
4-methoxy-2,3,6-trimethylbenzene sulphonyl (Mtr) and
9-fluorenylmethoxy-carbonyl (also designated Fmoc). It will be
appreciated that when the peptide is built up from the C-terminal
end, an amine-protecting group will be present on the .alpha.-amino
group of each new residue added and will need to be removed
selectively prior to the next coupling step.
[0109] Carboxyl protecting groups which may, for example be
employed include readily cleaved ester groups such as benzyl (Bzl),
p-nitrobenzyl (ONb), pentachlorophenyl (OPClP), pentafluorophenyl
(OPfp) or t-butyl (OtBu) groups as well as the coupling groups on
solid supports, for example methyl groups linked to
polystyrene.
[0110] Thiol protecting groups include p-methoxybenzyl (Mob),
trityl (Trt) and acetamidomethyl (Acm).
[0111] A wide range of procedures exists for removing amine- and
carboxyl-protecting groups. These must, however, be consistent with
the synthetic strategy employed. The side chain protecting groups
must be stable to the conditions used to remove the temporary
.alpha.-amino protecting group prior to the next coupling step.
[0112] Amine protecting groups such as Boc and carboxyl protecting
groups such as tBu may be removed simultaneously by acid treatment,
for example with trifluoroacetic acid. Thiol protecting groups such
as Trt may be removed selectively using an oxidation agent such as
iodine.
[0113] Peptides according to the invention may be prepared by
incomplete deprotection to leave groups which enhance the cytotoxic
activity of the peptides. Alternatively, modified R and N- and
C-terminal groups may be prepared after synthesis of the peptide
and associated deprotection.
[0114] A particularly preferred method involves synthesis using
amino acid derivatives of the following formula: Fmoc-amino
acid-Opfp.
[0115] References and techniques for synthesising peptidomimetic
compounds and the other bioactive molecules of the invention are
described herein and thus are well known in the art.
[0116] Formulations comprising one or more small bioactive
molecules as defined herein in admixture with a suitable diluent,
carrier or excipient constitute a further aspect of the present
invention. Such formulations may be for, inter alia, pharmaceutical
(including veterinary) or agricultural purposes or for use as
sterilising agents for materials susceptible to microbial
contamination, e.g. in the food industry. Suitable diluents,
excipients and carriers are known to the skilled man.
[0117] The peptides and other molecules defined herein exhibit
broad antimicrobial activity and thus are also suitable as
antiviral and antifungal agents, which will have pharmaceutical and
agricultural applications, and as promoters of wound healing or
spermicides. All of these uses constitute further aspects of the
invention.
[0118] Methods of treating or preventing bacterial, viral or fungal
infections or of treating tumours which comprises administration to
a human or animal patient one or more of the peptides,
peptidomimetics or other bioactive molecules as defined herein
constitute further aspects of the present invention.
[0119] The compositions according to the invention may be
presented, for example, in a form suitable for oral, nasal,
parenteral, intravenal, intratumoral or rectal administration.
[0120] As used herein, the term "pharmaceutical" includes
veterinary applications of the invention.
[0121] The active compounds defined herein may be presented in the
conventional pharmacological forms of administration, such as
tablets, coated tablets, nasal sprays, solutions, emulsions,
liposomes, powders, capsules or sustained release forms. The
peptides are particularly suitable for topical administration, e.g.
in the treatment of diabetic ulcers. Conventional pharmaceutical
excipients as well as the usual methods of production may be
employed for the preparation of these forms. Tablets may be
produced, for example, by mixing the active ingredient or
ingredients with known excipients, such as for example with
diluents, such as calcium carbonate, calcium phosphate or lactose,
disintegrants such as corn starch or alginic acid, binders such as
starch or gelatin, lubricants such as magnesium stearate or talcum,
and/or agents for obtaining sustained release, such as
carboxypolymethylene, carboxymethyl cellulose, cellulose acetate
phthalate, or polyvinylacetate.
[0122] The tablets may if desired consist of several layers. Coated
tablets may be produced by coating cores, obtained in a similar
manner to the tablets, with agents commonly used for tablet
coatings, for example, polyvinyl pyrrolidone or shellac, gum
arabic, talcum, titanium dioxide or sugar. In order to obtain
sustained release or to avoid incompatibilities, the core may
consist of several layers too. The tablet-coat may also consist of
several layers in order to obtain sustained release, in which case
the excipients mentioned above for tablets may be used.
[0123] Organ specific carrier systems may also be used.
[0124] Injection solutions may, for example, be produced in the
conventional manner, such as by the addition of preservation
agents, such as p-hydroxybenzoates, or stabilizers, such as EDTA.
The solutions are then filled into injection vials or ampoules.
[0125] Nasal sprays which are a preferred method of administration
may be formulated similarly in aqueous solution and packed into
spray containers either with an aerosol propellant or provided with
means for manual compression. Capsules containing one or several
active ingredients may be produced, for example, by mixing the
active ingredients with inert carriers, such as lactose or
sorbitol, and filling the mixture into gelatin capsules.
[0126] Suitable suppositories may, for example, be produced by
mixing the active ingredient or active ingredient combinations with
the conventional carriers envisaged for this purpose, such as
natural fats or polyethyleneglycol or derivatives thereof.
[0127] Dosage units containing the active molecules preferably
contain 0.1-10 mg, for example 1-5 mg of the antimicrobial agent.
The pharmaceutical compositions may additionally comprise further
active ingredients, including other cytotoxic agents such as other
antimicrobial peptides. Other active ingredients may include
different types of antibiotics, cytokines e.g. IFN-.gamma., TNF,
CSF and growth factors, immunomodulators, chemotherapeutics e.g.
cisplatin or antibodies.
[0128] The bioactive molecules, when used in topical compositions,
are generally present in an amount of at least 0.1%, by weight. In
most cases, it is not necessary to employ the peptide in an amount
greater than 1.0%, by weight.
[0129] In employing such compositions systemically (intra-muscular,
intravenous, intraperitoneal), the active molecule is present in an
amount to achieve a serum level of the bioactive molecule of at
least about 5 ug/ml. In general, the serum level need not exceed
500 ug/ml. A preferred serum level is about 100 ug/ml.
[0130] Such serum levels may be achieved by incorporating the
bioactive molecule in a composition to be administered systemically
at a dose of from 1 to about 10 mg/kg. In general, the molecule(s)
need not be administered at a dose exceeding 100 mg/kg.
[0131] Methods of treating environmental or agricultural sites or
products, as well as foodstuffs and sites of food production with
one or more of the bioactive molecules as defined herein to reduce
the numbers of viable bacteria present or limit bacterial growth or
reproduction constitute further aspects of the present
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0132] The invention will now be further described with reference
to the following non-limiting Examples and the figures in
which:
[0133] FIG. 1 is an electronmicrograph of normal (untreated) E.
coli, and
[0134] FIG. 2 is an electronmicrograph of E. coli treated with one
of the peptides described herein, WRWRWR [SEQ ID NO:1]. The treated
bacteria are void of cytoplasmic matter and their cell membranes
(as well as cell wall components) are destroyed, clearly indicating
a lytic mechanism.
EXAMPLES
[0135] The following experiments exemplify the principles discussed
above. For convenience, cationic amino acids are represented by
arginine and bulky and lipophilic amino acids by tryptophan,
tyrosine and tri-tert.-butyl tryptophan (super bulky and
lipophilic). It is clear that other residues with similar charge or
bulk and lipophilicity could be used in place of these amino acids.
N and C terminal modifying groups provide further bulky and
lipophilic groups.
[0136] The antimicrobial efficacy was determined as the minimum
inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC) both in .mu.g/ml for E. coli and S. aureus,
representative Gram-negative and Gram positive bacteria. The
cellular toxicity was determined as EC50 (amount of peptide
necessary for 50% lysis of erythrocytes).
[0137] MIC (Minimum Inhibitory Concentration) Tests
[0138] The bacterial strains used were: Escherichia coli ATCC
25922, Staphylococcus aureus ATCC 25923, MRSA ATCC 33591 and MRSE
ATCC 27626. All strains were stored at -70.degree. C. The bacteria
were grown in 2% Bacto Peptone water (Difco 1807-17-4). All tests
were performed with bacteria in mid-logarithmic growth phase.
Determination of the minimum inhibitory concentration (MIC) of the
peptides for bacterial strains were performed in 1% Bacto Peptone
water. A standard microdilution technique with an inoculum of
2.times.10.sup.6 CFU/ml was used. All assays were performed in
duplicate. Since the peptides are positively charged and therefore
could adhere to the plastic wells, we controlled the actual
concentration of the peptides in the solution by HPLC. There was no
difference between the concentration of the peptides before or
after adding the solution to the plastic wells. MBC tests were
performed in an analogous manner.
[0139] Hemolytic Assay
[0140] The hemolytic activities of the peptides were determined
using fresh human red blood cells. 8 ml blood was taken from a
healthy person. 4 ml blood was transferred to a polycarbonate tube
containing heparin to a final concentration of 10 U/ml, and the
remaining 4 ml blood was transferred to a glass tube containing
EDTA with final concentration of 15% EDTA. The erythrocytes were
isolated from heparin-treated blood by centrifugation in 1500 rpm
for 10 min and washed three times with phosphate-buffered saline
(PBS) to remove plasma and buffy coat. The cell pellet was
resuspended in PBS to make the final volume of 4 ml. The peptide
was diluted to a concentration of 2 mg/ml and 0.1 mg/ml. The
peptide was further diluted to the concentrations as stated in
Table 15. For each tube PBS was added first, then RBCs and peptide
solutions. The hematocrit in the blood treated with EDTA was
determined after 30 min with Sysmex K-1000, and the resuspended
RBCS were diluted into 10% hematocrit. RBCs in PBS (1%) with and
without peptides (Table 15) were incubated in a shaker at
37.degree. for 1 hour and then centrifuged at 4000 rpm for 5
min.
[0141] The supernatant were carefully transferred to new
polycarbonate tubes and the absorbance of the supernatant was
measured at 540 nm. Baseline hemolysis was hemoglobin released in
the presence of PBS, and 100% hemolysis was hemoglobin released in
the presence of 0.1% Triton X-100.
Example 1
[0142] A series of peptides was prepared on a solid phase multiple
peptide synthesizer MBS 396 from Advance Chemtech with Arg-Trp
combinations with C-terminal amidation to avoid negative charge
from the carboxylate. The antibacterial activity of these peptides
is shown in Table 1 below.
TABLE-US-00001 TABLE 1 Antibacterial activity of short RW and
similar peptide amides MIC MBC MIC MBC Sequence E. coli E. coli S.
aureus S. aureus WRWRWR 7.5 15 7.5 10 [SEQ ID NO: 1] RRRWWW 10 (20)
20 5 (<2.5) 10 (20) [SEQ ID NO: 2] RWWWRR 10 15 7.5 10 [SEQ ID
NO: 3] WWRRRW 20 (20) 20 10 (<2.5) 20 (25) [SEQ ID NO: 4] RWRWRW
10 (20) 20 5 (<2.5) 10 [SEQ ID NO: 5] RWRYRW 50 (10) 10
(<2.5) [SEQ ID NO: 6] WRWRW 20 (10) 50 5 (<2.5) 20 (20) [SEQ
ID NO: 7] WRYRW 75 (20) 50 (<2.5) [SEQ ID NO: 8] RWRWR 50 (20)
100 20 20 [SEQ ID NO: 9] WRWRY 75 (20) 50 (<2.5) [SEQ ID NO: 10]
RWWR >100 >100 10 >100 [SEQ ID NO: 11] WRRW >100
>100 75-100 >100 [SEQ ID NO: 12] WRWR >100 >100 100
>100 [SEQ ID NO: 13] WRW >100 >100 75 100 [SEQ ID NO: 14]
RWR >100 >100 >100 >100 [SEQ ID NO: 15]
[0143] The values in brackets refer to peptides in which one or
more of the tryptophan residues have been modified by the PMC
group.
Example 2
[0144] A second series of peptides was prepared manually by
synthesis in solution incorporating an Arg/Trp (Tbt) or Trp
(Tbt)/Arg motif with C-terminal esterification and/or N-terminal
acylation. The second set of peptides were designed on the basis of
preparing a small number of building blocks (i.e. Boc RW OBz, Boc
WE OMe and Boc TbtR OMe) and modifying these with additional amino
acids (at the N-terminus), N-terminal acylation and/or Cterminal
modification (preparation of a cationic site at C-terminus by
making a diamino ethane derivative).
[0145] General Procedure for the Removal of Boc
[0146] The Boc protected peptide was dissolved in reagent K.sup.i
and stirred at room temperature for 60-90 minutes..sup.1 To the
reaction mixture was added a solution of p-toluensulphonic acid
(2.0-2.5 eq) dissolved in a minimal amount of diethylether.sup.i
and the milky white mixture was cooled in the refrigerator
overnight to allow the product to completely precipitate. The ether
layer was drained off, and the residue triturated with diethylether
before evaporation in vacuo to a powder. The crude product was
purified by RP-HPLC prior to biological testing, or used in the
next step without further purification.
[0147] Boc-D-Arg-D-Trp-OBzl (KP-2-1)
[0148] To a stirred solution of Boc-D-Arg-OH hydrochloride (855 mg,
2.75 mmoles), H-D-Trp-OBzl hydrochloride (832 mg, 2.5 mmoles), HOBt
(1378 mg, 9 mmoles) and DIPEA (2.05 ml, 12 mmoles) in DMF (5 ml)
and dichloromethane (1 ml) cooled on ice was added HBTU (1138 mg, 3
mmoles) in small portions over 10 minutes. The mixture was stirred
on ice for 1 hour, 40 ml dichloromethane was added and the organic
phase washed successively with 3.times.40 ml saturated NaHCO.sub.3,
2.times.30 ml 5% citric acid, 50 ml water and 2.times.30 ml brine.
Evaporation afforded a white solid.
[0149] H-D-Arg-D-Trp-OBzl (KP-2-2-1)
[0150] Boc-D-Arg-D-Trp-OBzl (1.29 g, 2.2 mmoles) was treated with
reagent K as described in the general procedure. Evaporation after
removal of the etheral layer afforded 0.85 g of a yellowish
solid.
[0151] Boc-L-Arg-L-Trp-OBzl (KP-1-2)
[0152] To a stirred solution of Boc-L-Arg-OH (792 mg, 2.75 mmoles),
H-L-Trp-OBzl hydrochloride (832 mg, 2.5 mmoles), HOBt (1378 mg, 9
mmoles) and DIPEA (2.05 ml, 12 mmoles) in DMF (6 ml) and
dichloromethane (3 ml) cooled on ice was added HBTU (1138 mg, 3
mmoles) in small portions over 10 minutes. The mixture was stirred
on ice for 45 minutes and at room temperature for 45 minutes.
Workup was performed as described for KP-2-1. Evaporation afforded
1.7 g of a yellowish solid.
[0153] H-L-Arg-L-Trp-OBzl (KP-4-1)
[0154] Boc-L-Arg-L-Trp-OBzl (1.0 g, 1.5 mmoles) was treated with
reagent K as described in the general procedure. Evaporation after
removal of the etheral layer afforded 1.07 g of a beige solid.
[0155] Boc-L-Trp-L-Arg-OMe (KP-3-2)
[0156] To a stirred solution of Boc-L-Trp-OH (761 mg, 2.5 mmoles),
H-L-Arg-OMe dihydrochloride (718 mg, 2.75 mmoles), HOBt (1378 mg, 9
mmoles) and DIPEA (2.05 ml, 12 mmoles) in DMF (5 ml) and
dichloromethane (2 ml) cooled on ice was added HBTU (1138 mg, 3
mmoles) in small portions over 10 minutes. The mixture was stirred
on ice for 1 hour, 40 ml ethyl acetate was added and the organic
phase washed successively with 3.times.40 ml saturated NaHCO.sub.3,
2.times.30 ml 5% citric acid, 50 ml water and 2.times.30 ml brine.
Evaporation afforded 1.1 g of a white solid.
[0157] H-L-Trp-L-Arg-OMe (KP-5-1)
[0158] Boc-L-Trp-L-Arg-OMe (1.1 g, 2.15 mmoles) was treated with
reagent K as described in the general procedure. Evaporation after
removal of the etheral layer afforded 1.23 g of a yellowish
solid.
[0159] Boc-L-Trp-L-Trp-L-Arg-OMe (KP-6-1)
[0160] To a stirred solution of Boc-L-Trp-OH (87 mg, 0.29 mmoles),
H-L-Trp-L-Arg-OMe di-p-toluenesulphonic acid (226 mg, 0.3 mmoles),
HOBt (158 mg, 1.03 mmoles) and DIPEA (235 .mu.l, 1.37 mmoles) in
DMF (2 ml) cooled on ice was added HBTU (130 mg, 0.34 mmoles) in
small portions over 10 minutes. The mixture was stirred on ice for
80 minutes, 5 ml dichloromethane was added and the organic phase
washed successively with 3.times.5 ml saturated NaHCO.sub.3,
2.times.5 ml 5% citric acid, 5 ml water and 5 ml brine. Evaporation
afforded 0.05 g of a yellow oil which, as judged by Tlc, contained
only minor amounts of product. The pooled water phases were
extracted with 3.times.15 ml ethyl acetate, dried over MgSO.sub.4
and evaporated to afford 0.17 g of an yellow oil. This oil was used
in the next step without further purification.
[0161] H-L-Trp-L-Trp-L-Arg-OMe (KP-8-1)
[0162] Boc-L-Trp-L-Trp-L-Arg-OMe (0.14 g, ca 0.2 mmoles) was
treated with reagent K as described in the general procedure. The
product was precipitated by the addition of diethyl ether without
added p-toluenesulphonic acid.
[0163] Boc-L-Arg-L-Trp-L-Arg-OMe (KP-11-1)
[0164] To a stirred solution of Boc-L-Arg-OH (64 mg, 0.22 mmoles),
H-L-Trp-L-Arg-OMe di-p-toluenesulphonic acid (175 mg, 0.23 mmoles),
HOBt (128 mg, 0.83 mmoles) and DIPEA (181 .mu.l, 1.1 mmoles) in DMF
(1 ml) cooled on ice was added HBTU (106 mg, 0.28 mmoles) in small
portions over 10 minutes. The mixture was stirred on ice for 2
hours and at room temperature for 30 minutes, 10 ml ethyl acetate
was added and the organic phase washed as described for KP-3-2.
After workup, the organic layer contained no amount of the desired
product as judged by analytical RP-HPLC. The pooled water phases
were extracted with 3.times.15 ml ethyl acetete and evaporated to
afford 0.1 g of an yellow oil. This oil was used in the next step
without further purification.
[0165] H-L-Arg-L-Trp-L-Arg-OMe (KP-13-1)
[0166] Boc-L-Trp-L-Trp-L-Arg-OMe (0.14 g, ca 0.2 mmoles) was
treated with reagent K as described in the general procedure. The
product was precipitated by the addition of diethyl ether without
added p-toluenesulphonic acid. Evaporation after removal of the
etheral layer afforded 0.1 g of a white solid.
[0167] Boc-L-Trp-L-Arg-L-Trp-OBzl (KP-12-1)
[0168] To a stirred solution of Boc-L-Trp-OH (88 mg, 0.29 mmoles),
H-L-Arg-L-Trp-OBzl di-p-toluenesulphonic acid (255 mg, 0.3 mmoles),
HOBt (158 mg, 1.03 mmoles) and DIPEA (235 .mu.l, 1.37 mmoles) in
DMF (2 ml) cooled on ice was added HBTU (130 mg, 0.34 mmoles) in
small portions over 10 minutes. The mixture was stirred on ice for
2 hours and at room temperature for 30 minutes, 10 ml ethyl acetate
was added and the organic phase washed as described for KP-3-2.
Evaporation afforded 0.23 g of a yellow oil.
[0169] H-L-Trp-L-Arg-L-Trp-OBzl (KP-14-1)
[0170] Boc-L-Trp-L-Arg-L-Trp-OBzl (0.23 g, ca 0.3 mmoles) was
treated with reagent K as described for KP-8-1. Evaporation after
removal of the etheral layer afforded 0.2 g of a white solid.
[0171] Boc-D-Trp-L-Arg-L-Trp-OBzl (KP-15-1)
[0172] To a stirred solution of Boc-D-Trp-OH (90 mg, 0.29 mmoles),
H-L-Arg-L-Trp-OBzl di-p-toluenesulphonic acid (251 mg, 0.3 mmoles),
HOBt (158 mg, 1.03 mmoles) and DIPEA (235 .mu.l, 1.37 mmoles) in
DMF (5 ml) cooled on ice was added HBTU (130 mg, 0.34 mmoles) in
small portions over 10 minutes. The mixture was stirred on ice for
40 minutes, and workup performed as described for KP-2-1.
Evaporation afforded 0.24 g of a white solid.
[0173] H-D-Trp-L-Arg-L-Trp-OBzl (KP-16-1)
[0174] Boc-Trp-Arg-Trp-OBzl (0.24 g, ca 0.3 mmoles) was treated
with reagent K as described in the general procedure. Evaporation
after removal of the etheral layer afforded 0.17 g of a beige
solid.
[0175] Boc-D-Trp-D-Arg-D-Trp-OBzl (KP-2-1-2)
[0176] To a stirred solution of Boc-D-Trp-OH (162 mg, 0.53 mmoles),
H-D-Arg-D-Trp-OBzl di-p-toluenesulphonic acid (464 mg, 0.56
mmoles), HOBt (292 mg, 1.9 mmoles) and DIPEA (4.4 ml, 25 mmoles) in
DMF (5 ml) and dichloromethane (1 ml) cooled on ice was added HBTU
(241 mg, 0.64 mmoles) in small portions over 10 minutes. The
mixture was stirred on ice for 70 minutes, 10 ml dichloromethane
was added and the organic phase washed successively with 3.times.10
ml saturated NaHCO.sub.3, 4.times.10 ml 5% citric acid (until
acidic water phase due to too much DIPEA added), 2.times.10 ml
water and 2.times.10 ml brine. Evaporation afforded 0.42 g crude
product.
[0177] H-D-Trp-D-Arg-D-Trp-OBzl (KP-2-1-3)
[0178] Boc-D-Trp-D-Arg-D-Trp-OBzl (0.38 g, ca 0.4 mmoles) was
treated with reagent K as described in the general procedure.
Evaporation after removal of the etheral layer afforded 0.3 g of a
beige solid.
[0179] Boc-L-Arg-L-Trp-OH (KP-10-2)
[0180] To a solution of Boc-L-Arg-L-Trp-OH (300 mg, 0.5 mmoles) in
5 ml methanol/water (19:1) Pd-10% on charcoal (53 mg, 0.05 mmoles)
was added. The mixture was stirred under a hydrogen atmosphere (1
atm) overnight, filtered through a thin layer of Celite 545 and
evaporated to afford a red oil. The oil was dissolved in water
under gentle heating and lyophilized to afford 383 mg of a pink
powder.
[0181] Boc-L-Arg-L-Trp-L-Arg-L-Trp-OBzl (KP-17-1) [SEQ ID
NO:16]
[0182] To a stirred solution of Boc-L-Arg-L-Trp-OH hydrochloride
(100 mg, 0.20 mmoles), H-L-Arg-L-Trp-OBzl di-p-toluenesulphonic
acid (175 mg, 0.21 mmoles), HOBt (110 mg, 0.72 mmoles) and DIPEA
(164 .mu.l, 0.96 mmoles) in DMF (2 ml) cooled on ice was added HBTU
(91 mg, 0.24 mmoles) in small portions over 10 minutes. The mixture
was stirred on ice for 3 hours and workup performed as described
for KP-3-2.
[0183] H-L-Arg-L-Trp-L-Arg-L-Trp-OBzl (KP-19-1) [SEQ ID NO:17]
[0184] Boc-L-Arg-L-Trp-L-Arg-L-Trp-OBzl (ca 0.2 mmoles) was treated
with reagent K as described for KP-8-1. Complete removal of the
etheral layer was difficult to perform without loss of material and
the pink crude product therefore probably contained significant
amounts of TFA.
[0185] Boc-L-Arg-L-Trp-D-Arg-D-Trp-OBzl (KP-18-1) [SEQ ID
NO:18]
[0186] To a stirred solution of Boc-L-Arg-L-Trp-OH hydrochloride
(100 mg, 0.20 mmoles), H-D-Arg-D-Trp-OBzl di-p-toluenesulphonic
acid (175 mg, 0.21 mmoles), HOBt (110 mg, 0.72 mmoles) and DIPEA
(164 .mu.l, 0.96 mmoles) in DMF (2 ml) cooled on ice was added HBTU
(91 mg, 0.24 mmoles) in small portions over 10 minutes. The mixture
was stirred on ice for 3 hours and workup performed as described
for KP-3-2. After workup, the organic layer contained only minor
amounts of the desired product as judged by analytical RP-HPLC. The
pooled water phases was extracted with 3.times.15 ml ethyl acetete
and evaporated to afford the crude product.
[0187] H-L-Arg-L-Trp-D-Arg-D-Trp-OBzl (KP-20-1) [SEQ ID NO:19]
[0188] Boc-L-Arg-L-Trp-D-Arg-D-Trp-OBzl (ca 0.2 mmoles) was treated
with reagent K as described for KP-8-1. Complete removal of the
etheral layer was difficult to perform without loss of material and
the crude product therefore probably contained significant amounts
of TFA.
[0189] Boc-L-Arg-L-Trp-L-Trp-L-Arg-OMe (KP-21-1) [SEQ ID NO:20]
[0190] To a stirred solution of Boc-L-Arg-L-Trp-OH hydrochloride
(100 mg, 0.20 mmoles), H-L-Trp-L-Arg-OMe di-p-toluenesulphonic acid
(171 mg, 0.23 mmoles), HOBt (110 mg, 0.72 mmoles) and DIPEA (164
.mu.l, 0.96 mmoles) in DMF (2 ml) cooled on ice was added HBTU (91
mg, 0.24 mmoles) in small portions over 10 minutes. The mixture was
stirred on ice for 3 hours and workup performed as described for
KP-3-2. After workup, the organic layer contained only minor
amounts of the desired product as judged by analytical RP-HPLC. The
pooled water phases were extracted with 3.times.15 ml ethyl acetete
and evaporated to afford 0.16 g of a yellow oil.
[0191] H-L-Arg-L-Trp-L-Trp-L-Arg-OMe (KP-22-1) [SEQ ID NO:21]
[0192] Boc-L-Arg-L-Trp-L-Trp-L-Arg-OMe (0.16 g, ca 0.18 mmoles) was
treated with reagent K as described for KP-8-1. Evaporation after
removal of the etheral layer afforded 0.12 g of a pink solid.
[0193] Ind-Trp-Arg-OMe
[0194] 3-Indolylacetic acid (0.289 mmoles) was treated with
H-Trp-Arg-OMe (1.06 eq), triethylamine (2.01 eq) and HBTU (1.10 eq)
as described for Boc-Trp-Arg-OMe. Methanol was used as solvent. The
reaction was quenched by adding 9 ml saturated sodium chloride. The
aqueous phase was extracted 3.times.7 ml ethyl acetate and the
organic phase washed with 4 ml 2 M hydrochlorid acid, 4 ml water
and 4 ml 5% sodium hydrogen carbonate. The washing procedure was
repeated one time before the organic phase was dried with 5 ml
saturated sodium chloride and then evaporated to yield 0.11 g of a
white solid. The crude product was purified by RP-HPLC.
[0195] .sup.1H NMR (acetonitril-d.sub.3): .delta.=1.34 (2H, m),
1.52 (1H, m), 1.71 (1H, m), 2.89 (5H, m), 3.05 (1H, m), 3.15 (1H,
m), 4.34 (1H, m), 4.48 (1H, m), 6.01 (4H, bs), 6.42 (1H, bs),
6.88-7.50 (12H, m' s), 9.10 (1H, s), 9.22 (1H, s).
[0196] Chx-Trp-Arg-OMe
[0197] Cyclohexane carboxylic acid (0.297 mmoles) was treated with
H-Trp-Arg-OMe (1.03 eq), triethylamine (1.96 eq) and HBTU (1.05 eq)
as described for Boc-Trp-Arg-OMe. Methanol was used as solvent.
Quenching and work up was performed as for Ind-Trp-Arg-OMe to yield
0.10 g of a white solid. The crude product was purified by
RP-HPLC.
[0198] Boc-Tbt-Arg-OMe
[0199] A stirred solution of Boc-Tbt-OH (0.4735 g, 1.0 mmole),
H-Arg-OMe dihydrochloride (0.2741 g, 1.05 mmoles), HOBt (0.4872 g,
3.61 mmoles) and DIPEA (0.820 ml, 4.79 mmoles) in
DMF/dichloromethane (14 ml) is cooled in an ice/water bath. HBTU
(0.4560 g, 1.2 mmoles) is added in small portions over 10 min. The
mixture is stirred for 30 min and the cooling bath removed. The
reaction mixture is allowed to stir at room temperature until no
carboxylic acid component is left (Tlc system A). The mixture is
evaporated to an oil, 20 ml dichloromethane is added and the
organic phase washed 3.times.20 ml saturated sodium hydrogen
carbonate, 2.times.15 ml 5% citric acid, 25 ml water and 2.times.15
ml saturated sodium chloride successively, and evaporated to give
0.80 g of a white solid.
[0200] H-Tbt-Arg-OMe
[0201] Boc-Tbt-Arg-OMe (0.90 mmoles) was treated with reagent K as
described in the general method. Evaporation after removal of the
etheral layer afforded 0.24 g of a white solid. The crude product
was purified by RP-HPLC.
[0202] Boc-Arg-Tbt-Arg-OMe
[0203] The di-p-toluensulfonic acid salt of H-Tbt-Arg-OMe (0.24 g,
0.270 mmoles), Boc-Arg-OH (0.0933 g, 0.335 mmoles), HOBt (0.0442 g,
0.327 mmoles), triethylamine (0.113 ml, 0.811 mmoles) and HBTU
(0.1231 g, 0.325 mmoles) were dissolved in acetonitrile (2.2 ml,
HPLC-grade) and stirred at room temperature. After 1 hr starting
material was still left (Tlc system A). Three equivalents of
triethylamine were added and the reaction mixture stirred for
another hr. Quenching and workup was performed as described for
Boc-Trp-Arg-OMe. The crude oil was coevaporated with
dichloromethane to afford 0.23 g of a white powder. The crude
product was used without further purifications.
[0204] H-Arg-Tbt-Arg-OMe
[0205] The crude Boc-Arg-Tbt-Arg-OMe was dissolved in 4.05 ml of
reagent K and the cleavage performed as described in the general
procedure. The crude product was purified by RP-HPLC prior to
biological testing.
[0206] Boc-Arg-Trp-OBzl
[0207] Boc-Arg-OH and H-Trp-OBzl coupled as described for
Boc-Trp-Arg-OMe. N-methyl morpholine used as base. The crude
product was purified by RP-HPLC.
[0208] H-Arg-Trp-OBzl
[0209] The crude Boc-Arg-Trp-OBzl (1.23 mmoles) was dissolved in
18.3 ml of reagent K and the cleavage performed as described in the
general procedure. The crude product was used without further
purification.
[0210] Ind-Arg-Trp-OBzl
[0211] 3-Indolylacetic acid (0.0450 g, 0.257 mmoles) was treated
with H-Arg-Trp-OBzl di-p-toluensulphonic acid salt, HBTU and
triethylamine (6 eq) as described for Boc-Trp-Arg-OMe. Acetonitrile
was used as solvent. The crude product was isolated as 0.19 g of a
white solid.
[0212] Chx-Arg-Trp-OBzl
[0213] Cyclohexane carboxylic acid (0.0031 ml, 0.266 mmoles) was
treated with H-Arg-Trp-OBzl di-p-toluensulphonic acid salt, HBTU
and triethylamine (6 eq) as described for Boc-Trp-Arg-OMe.
Acetonitrile was used as solvent. The crude product was isolated as
0.17 g of a white solid.
[0214] Ind-Arg-Trp-OH
[0215] The crude Ind-Arg-Trp-OBzl was hydrogenated as described in
the general method to afford an yellow oil.
[0216] Chx-Arg-Trp-OH
[0217] The crude Chx-Arg-Trp-OBzl was hydrogenated as described in
the general method to afford an yellow oil.
[0218] Boc-Arg-Trp-OH (B87)
[0219] The crude Boc-Arg-Trp-OBzl was hydrogenated as described in
the general method to afford 0.72 g of an yellow oil.
[0220] Boc-Arg-Trp-Dae-Z
[0221] To a stirred solution of Boc-Arg-Trp-OH (1.265 mmoles) in
DMF/dichloromethane (12 ml, 1:1), HOBt (0.6202 g, 4.590 mmoles),
DIPEA (1.040 ml, 6.075 mmoles) and N--Z-diaminoethane hydrochloride
(0.3088 g, 1.339 mmoles) were added. HBTU (0.5772 g, 1.522 mmoles)
was added in small portions over 5 min. The reaction mixture was
stirred at room temperature for 3 hrs and 45 min and evaporated to
a dark yellow oil. The oil was dissolved in 20 ml ethylacetate and
washed with 3 ml 2 M hydrochloric acid, 5 ml water 5 ml 5% sodium
hydrogen carbonate and 5 ml water successively. The resulting dark
yellow solution was dried over magnesium sulphate and evaporated to
an oil. Trituration in heptane failed, and the oil was evaporated
to yield 0.86 g of a brownish solid.
[0222] H-Arg-Trp-Dae-Z
[0223] Boc-Arg-Trp-Dae-Z (0.544 mmoles) was dissolved in reagent K
(6.8 ml TFA) and stirred at room temperature for 1 hr and 10
minutes. The reaction mixture was evaporated to a small volume and
a solution of p-toluensulphonic acid (0.32 g) in diethylether (20
ml) was added. The milky white mixture was cooled in the
refrigerator overnight to allow the product to completely
precipitate. The ether layer was drained off, and the residue
evaporated in vacuo to afford a white powder. The crude product was
purified by RP-HPLC.
[0224] Ind-Arg-Trp-Dae-Z
[0225] 3-Indolylacetic acid (0.0265 g, 0.153 mmoles) was treated
with H-Arg-Trp-Dae-Z di-p-toluensulphonic acid salt, HBTU (1.2 eq)
and triethylamine (5 eq) as described for Boc-Trp-Arg-OMe.
Acetonitrile (1.4 ml) and DMF (0.6 ml) were used as solvents, due
to poor solubility of the dipeptide analog in acetonitrile. The
crude product was isolated as 0.12 g of a white solid.
[0226] Abbreviations [0227] Arg arginine [0228] Boc
t-butyloxycarbonyl [0229] Chx cyclohexane carboxylic acid [0230]
Dae diamino ethane [0231] DIPEA diisopropylethylamine [0232] DMF
N,N'-dimethylformamide [0233] ESMS Electrospray Mass Spectrometry
[0234] HBTU O-(Benzotriazol-1-yl)N,N,N',N'-tetramethyluronium
hexafluorophosphate [0235] HOBt 1-hydroxybenzotriazole [0236] Ind
3-indolylacetic acid [0237] MBC Minimum Bactericidal Concentration
[0238] MIC Minimum Inhibitory Concentration [0239] RP-HPLC Reversed
Phase High Performance Liquid Chromatography [0240] Tbt
2,5,7-tri-t-butyl tryptophan [0241] TFA Trifluoroacetic acid [0242]
Trp tryptophan [0243] Z benzyloxycarbonyl
REFERENCES
[0243] [0244] 1) Guy, C. A.; Fields, G. B. Methods in enzymology
1997, 289, 67-83. [0245] 2) Lott, R. S.; Chauhan, V. S.; Stammer,
C. H. Journal of the Chemical Society Chemical Communications 1979,
495-496.
[0246] Notes
[0247] Reagent K consists of phenol (5%, w/v), water (5% v/v),
thioanisole (5% v/v), ethanedithiol (2. % v/v) and trifluoroacetic
acid (82.5% v/v). 1.5 ml of the reagent per mmoles of the peptide
is used for cleavage of the Boc group.
[0248] The addition of p-toluenesulphonic acid in diethyl ether
results in the formation of the p-toluenesulphonic acid salts of
the peptides. Peptides containing one free amino function or a
guanidine function are believed to form the mono p-toluenesulphonic
acid salt etc.
[0249] Identification of the products was performed using ESMS and
the results are shown in Table 2.
TABLE-US-00002 TABLE 2 Analytical results for peptides
Sequence.sup.a Code MW ESMS Purity RW-OBzl KP-4-1/1 450.53 451.1
97% [SEQ ID NO: 22] rw-OBzl KP-2-2-1 450.53 451.1 98% [SEQ ID NO:
23] WR-OMe KP-5-1/1 374.44 375.2 97% [SEQ ID NO: 24] WRW-OBzl
KP-14-1/1 636.73 637.4 98% [SEQ ID NO: 25] wrw-OBzl KP-2-1-3 636.73
637.3 97% [SEQ ID NO: 26] wRW-OBzl KP-16-1/1 636.73 637.4 98% [SEQ
ID NO: 27] WWR-OMe KP-8-1/1A 560.64 561.4 97% [SEQ ID NO: 28]
RWR-OMe KP-13-1/1 560.64 531.4 98% [SEQ ID NO: 29] RWRW-OBzl
KP-19-1/2u 792.93 793.4 97% [SEQ ID NO: 30] RWrw-OBzl KP-20-1/1
792.93 793.4 98% [SEQ ID NO: 31] RWWR-OMe KP-22-1 716.84 717.4 99%
[SEQ ID NO: 32] .sup.aCapital letters represent L-amino acids,
non-capital letters represent D-amino acids
[0250] The chemical yield of the coupling and deprotection
reactions has not been measured. Identification of the products has
been done using ESMS. Purification has been performed with RP-HPLC
on a semi-preparative C18 coloumn with water and acetonitrile (both
added 0.01% TFA) as mobile phase. Peptide content in the purified
samples has been established with RP-HPLC on an analytical C18
coloumn.
[0251] The antibacterial activity of these peptides is shown in
Tables 3-5 below.
TABLE-US-00003 TABLE 3 Antibacterial activity of short peptide
derivatives.sup.a MIC MIC MIC MIC Sequence.sup.b E. coli S. aureus
MRSA MRSE RW-OBzl >200 >200 50 50 25/50 20 20 20 [SEQ ID NO:
22] rw-OBzl >200 >200 50 50 50/75 50 20 20 [SEQ ID NO: 23]
WR-OMe >200 >200 >200 =200 >200 >200 >200 >200
[SEQ ID NO: 24] WRW-OBzl 75 75 5 5 5 2.5 5 5 [SEQ ID NO: 25]
wrw-OBzl 100 50 5 5 5 5 5 5 [SEQ ID NO: 26] wRW-OBzl 75 75/100 20
20 20 10 10 10 [SEQ ID NO: 27] WWR-OMe >200 >200 >200 =200
200 200 200 200 [SEQ ID NO: 28] RWR-OMe >200 >200 >200
>200 200 >200 100 100 [SEQ ID NO: 29] RWRW-OBzl 75/100 75 5 5
5 5 2.5 2.5 [SEQ ID NO: 30] RWrw-OBzl 75 50/75 5 5 5 2.5 2.5 --
[SEQ ID NO: 31] RWWR-OMe >200 >200 75 75 50 50 20 20 [SEQ ID
NO: 32] .sup.aConcentration series: 200, 100, 75, 50, 25, 10, 5 and
2.5 .mu.g/ml. .sup.bCapital letters represent L-amino acids,
non-capital letters represent D-amino acids MIC values in .mu.g/ml
E. coli S. aureus MRSA MRSE Rw-OBzl >300 37.5 37.5 25 rW-Obzl
>300 37.5 50 25 WR-OBzl >300 100 37.5 KW-Obzl >300 37.5
37.5 25 kW-Obzl >300 100 RF-Obzl >300 300 150 100 FR-Obzl
>300 >300 37.5 100 KF-Obzl >300 300 300 150 Concentration
series: 300, 100, 50, 37.5 and 25 .mu.g/ml Capital Letters
represent L-amino acids, non-capital letters represent D-amino
acids
TABLE-US-00004 TABLE 4 Antibacterial activity of short peptide
derivatives.sup.a MIC MIC MIC MIC Sequence.sup.b E. coli S. aureus
MRSA MRSE RW-OBzl >200 >200 50 50 75 50 20 20 [SEQ ID NO: 22]
rw-OBzl >200 >200 75 100 100 75 25/50 25 [SEQ ID NO: 23]
WR-OMe >200 >200 >200 >200 >200 >200 >200
>200 [SEQ ID NO: 24] WRW-OBzl 200 200 5 5 10 10/20 10 5 [SEQ ID
NO: 25] wrw-OBzl 200 200 5 10 10 10 5 5 [SEQ ID NO: 26] wRW-OBzl
200 100 20 20 20 10 10 10 [SEQ ID NO: 27] WWR-OMe >200 >200
>200 >200 200 200 200 200 [SEQ ID NO: 28] RWR-OMe >200
>200 >200 >200 >200 >200 200 200 [SEQ ID NO: 29]
RWRW- 100 100 10 5 10 5 2.5/5 2.5 OBzl [SEQ ID NO: 30] RWrw-OBzl 75
100 5 5 5 5 2.5 -- [SEQ ID NO: 31] RWWR- >200 >200 =200
100/200 100 200 20 20 OMe [SEQ ID NO: 32] .sup.aConcentration
series: 200, 100, 75, 50, 25, 10, 5 and 2.5 .mu.g/ml. .sup.bCapital
letters represent L-amino acids, non-capital letters represent
D-amino acids
TABLE-US-00005 TABLE 5 Antibacterial activity of short peptide
derivatives Values are given as MIC (MBC) values in .mu.g/ml.
Peptide.sup.a E. coli S. aureus MRSA.sup.b MRSE.sup.c Dipeptides RW
OBz >200 (>200) 50 (50) 25 (50) 20 (20) rw OBz >200
(>200) 50 (75) 50 (75) 20 (25) RW DaeZ >200 (>200) 50 (75)
Ind RW OBz >200 (>200) 20 (20) Chx RW OBz 200 (>200) 75
(75) Ind RW DaeZ >200 (>200) 75 (75) WR OMe >200 (>200)
>200 (>200) >200 (>200) >200 (>200) Ind WR OMe
>200 (>200) >200 (>200) Chx WR OMe >200 (>200)
>200 (>200) Tripeptides WRW OBz 75 (200) 5 (5) 5 (10) 5 (5)
wrw OBz 75 (200) 5 (5) 5 (10) 5 (5) wRW OBz 75 (100) 20 (20) 20
(20) 10 (10) WWR OMe >200 (>200) >200 (>200) >200
(>200) >200 (>200) RWR OMe >200 (>200) >200
(>200) >200 (>200) 100 (200) Tetrapeptides RWRW OBz 75
(100) 5 (5-10) 5 (5-10) 2.5 (2.5) [SEQ ID NO: 33] RWrwOBz 75
(75-100) 5 (5) 2.5-5 (5) 2.5 (2.5) [SEQ ID NO: 34] RWWR OMe >200
(>200) 75 (100-200) 50 (100-200) 20 (20) [SEQ ID NO: 32] Super
bulky TbtR OMe 25 (200) 10 (10) RTbtR OMe 25 (50) 5 (5)
.sup.aCapital letters represent L-amino acids, non-capital letters
represent D-amino acids. .sup.bMRSA is Methicillin resistant S.
aureus. .sup.cMRSE is Methicillin resistant S. epidermidis. Titer
series: 200, 100, 75, 50, 25, 10, 5, 2.5 .mu.g/ml
[0252] With the exception of the two peptides containing Tbt, none
of the peptides of Examples 1 or 2 displayed measurable haemolysis
(i.e. EC50 >1000 .mu.g/ml). The dipeptide Tbt-Arg-OMe had an
EC50 of 360 .mu.g/ml but surprisingly the tripeptide
Arg-Tbt-Arg-OMe was less toxic with an EC50 of 720 .mu.g/ml,
despite its higher activity against both E. coli and S. aureus.
[0253] Although arginine is preferred, lysine can be used without
significant loss of antibacterial activity. Phenylalanine, due to
its smaller size is less active than tryptophan.
Example 3
[0254] The peptide Arg-(2-Nal)-Arg-Tyr-Arg-(2-Nal)NH.sub.2 [SEQ ID
NO:35] wherein (2-Nal) is 2-naphtylalanine was prepared and tested
against a range of clinically important pathogens as shown in Table
6 below.
[0255] The peptide was synthesised on a 9050 Millipore Automatic
Peptide Synthesizer using Fmoc protection and activation with
pentafluorophenyl (Pfp)esters or in situ activation with the
coupling reagent HATU
(O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium
hexafluorophosphate). In the case of coupling with
pentafluorophenyl esters, 1-HOBt (1-hydroxybenzotriazole) was added
to catalyse the reaction, and when using the coupling reagent HATU
the reaction was base catalysed with DIPEA (diisopropylethylamine).
All amino acids with reactive side chains were protected with acid
labile protecting groups and cleaved upon treatment with TFA
(trifluoroacetic acid) containing scavengers. (See below for
scavenger mixture). At the same time the peptide was cleaved from
the solid support on treatment with the TFA solution.
[0256] A) Attachment of the first amino acid to the solid
support
[0257] The solid support PAC-PEG-PS (Peptide Acid-Poly Ethylene
Glycol-Poly Styrene resin) (1 eq.) was mixed together with
Fmoc-(2-Nal)-OPfp (5 eq.) and DMAP (dimethylaminopyridine) (1 eq.)
in a small volume of DMF (dimethylformamide) and left to swell for
30 minutes. The solution was then stirred slowly for 41/2 hours.
Ac.sub.2O (acetic acid anhydride) (2.5 eq.) and DMAP (0.1 eq.) were
then added to the solution in order to acetylate any remaining
hydroxyl groups on the solid support. The solution was then stirred
for another hour. The solid support with the C-terminai amino acid
attached was isolated by filtration and washed several times on the
filter with DMF. The solid support was then used in the synthesis
of the target peptide on the 9050 Millipore Automatic Peptide
Synthesizer.
[0258] B) Ninhydrin Test/Kaiser's Test
[0259] Less than 1 mg of the peptide-resin complex was treated with
small equal volumes of a 5% ninhydrin solution in ethanol, a
solution of 80 g phenol in 20 ml ethanol and a solution of dried,
distilled pyridine. The reaction mixture was heated for two minutes
at 110.degree. C., and investigated under a microscope. (In this
test a yellow reaction mixture indicates successful acetylation,
while a blue solution indicates still free amino groups.)
[0260] C) Cleavage of Acid Labile Protecting Groups
[0261] Cleavage of acid labile protection groups and cleavage of
the peptides from the solid support was achieved using a mixture of
2% anisol, 2% ethandithiol (EDT), 2% water and 2% phenol in TFA,
and with cleavage times of no more than four hours. The solid
support was then removed by filtration and the peptide precipitated
in diethyl ether. The ether solution containing TFA was removed
using a pasteur pipette, and the peptide was washed several times
with diethylether and dried under high vacuum.
[0262] D) Purification
[0263] The peptide was purified by HPLC using a C18-reversed phase
column (*) and a mixture of water and acetonitrile (both added 0.1%
TFA) as mobile phase. Selected wavelength for detection of peptide
fractions was 254 nm.
[0264] (*) PrePak.RTM.Cartridge 25.times.100 mm. DeltaPak.TM. C18
15 .mu.m 100 .ANG.. (waters corporation.)
[0265] E) Analysis
[0266] The peptide was analysed for impurities on an analytical
HPLC C18-reversed phase column using a mixture of water and
acetonitrile (both added 0.1% TFA) as mobile phase. The molecular
weight of the peptides were determined by positive ion electrospray
ionization mass spectrometry (VG Quattro Quadrupole).
[0267] Amino acid derivatives used in synthesis selected from the
following:
TABLE-US-00006 Fmoc-AlaPEG-PS (solid support) Fmoc-Lys(tBoc)-OPfp
Fmoc-Arg(Pbf)-OH Fmoc-Met-OPfp Fmoc-Arg(Pmc)-OH
Fmoc-.beta.-(2-naphthyl)-alanine-OH Fmoc-Asn(Trt)-OPfp
Fmoc-Phe-OPfp Fmoc-Cys(Acm)-OPfp Fmoc-Ser(tBu)-OPfp Fmoc-Gln-OPfp
Fmoc-Thr(tBu)-OPfp Fmoc-Glu(OtBu)-OPfp Fmoc-Trp-OPfp
Fmoc-Gly-OPfpFmoc-Tyr(tBu)-OPfp Fmoc-Leu-OPfp Fmoc-(2-Nal)-OPfp
[0268] Amino acid derivatives were purchased from either Bachem,
MilliGen/Biosearch (Division of Millipore) or PerSeptive
Biosystems.
TABLE-US-00007 TABLE 6 MIC MBC Pathogen (.mu.g/ml) (.mu.g/ml) E.
coli 10 15 S. aureus 5 5 MRSA 2.5 5 MRSE 2.5 2.5 MRSA = Methicillin
resistant S. aureus MRSE = Methicillin resistant S. epidermidis
Example 4
[0269] A further series of peptides was designed and made to
investigate the impact of different sized bulky and lipophilic
groups and their relative position with the molecule.
[0270] Most of the following peptides were made from the same
starting material, ROBzl. A method was developed for the
manufacture of ROBzl from BocR by the following 2 step method:
[0271] From the RoBzl starting material, the peptides were made
using the standard two step protocol with amide bond formation and
deprotection of the N-terminus.
[0272] In order to test the `super bulky` group, i.e. peptides
having only one very large bulky and lipophilic group, dipeptide
methyl esters (XROMe) were prepared. By way of example, the
synthesis of TbtR OMe is described in Example 2 above. Analogous
methods were used in the preparation of the other methyl
esters.
[0273] The antibacterial activity of the various peptides measured
as MIC in .mu.g/ml is shown in Table 7 below.
TABLE-US-00008 TABLE 7 Antibacterial activity measured as MIC in
.mu.g/ml. S. E. coli aureus MRSA MRSE P. aerug. Class Peptide
.mu.g/ml .mu.g/ml .mu.g/ml .mu.g/ml .mu.g/ml OBzl tBuGR-OBzl
>300.0 >300.0 100.0 150.0 >300.0 tBuAR-OBzl >300.0
300.0 150-200 100.0 >300.0 ChxAR- 300.0 50-100 25-37.5 25.0
>300.0 OBzl FR-OBzl >300.0 >300.0 25-37.5 100.0 >300.0
RF-OBzl >300.0 300.0 150.0 100.0 WR-OBzl >300.0 100.0 37.5
50.0 >300.0 RW-OBzl >300.0 100.0 50.0 25.0 tBuFR-OBzl 200.0
25.0 12.5-25 5.0 100.0 BipR-OBzl 150.0 5.0 5.0 5.0 100.0 OMe WR-OMe
>500.0 >500.0 >500.0 >500.0 RW-OMe >300.0 >300.0
>300.0 200.0 tBuFR-OMe >300.0 >300.0 100.0 100.0 >300.0
BipR-OMe >300.0 150.0 50.0 50.0 >300.0 TbtROMe 30.0 4.0 4.0
4.0 Titre series: 500, 300, 200, 150, 100, 50, 50, 37.5, 30, 25,
12.5, 5, 4, 2 and 1.
[0274] None of these peptides had measurable toxicity against red
blood cells.
Example 5
[0275] A further group of hexapeptides and tetrapeptides were
prepared on a solid phase multiple peptide synthesizer MBS 396 as
described in previous examples. These were tested against E. coli
and S. aureus and their minimum inhibitory concentrations (MIC) are
given in Table 8 below. The first column is the value in .mu.g/ml
and the second in .mu.M/ml. Alanine residues are included as
`spacers` and to investigate the impact of length on activity.
TABLE-US-00009 TABLE 8 Peptide E. coli S. aureus AAWWRR-NH2 Pmc+ 20
18.0 2.5 2.3 [SEQ ID NO: 36] RRAAWW-NH2 Pmc+ 20 18.0 2.5 2.3 [SEQ
ID NO: 37] AWRWRA-NH2 Pmc+ 20 18.0 2.5 2.3 [SEQ ID NO: 38]
WRAAWR-NH2 Pmc+ 50 45.0 2.5 2.3 [SEQ ID NO: 39] WWAARR-NH2 Pmc+ 50
45.0 5 4.5 [SEQ ID NO: 40] WWAARR-NH2 >200 237.0 >200 237.0
[SEQ ID NO: 41] AAWWRR-NH2 >200 237.0 >200 237.0 [SEQ ID NO:
42] RRAAWW-NH2 >200 237.0 >200 237.0 [SEQ ID NO: 43]
WRAAWR-NH2 >200 237.0 >200 237.0 [SEQ ID NO: 44] >200
237.0 >200 237.0 AWRWRA-NH2 [SEQ ID NO: 45] BBRR-NH2 50 64.4 5
6.4 [SEQ ID NO: 46] WBRR-NH2 Pmc+ >100 99.5 5-10 5.0-9.9 [SEQ ID
NO: 47] WBRR-NH2 >100 135.3 20 27.1 [SEQ ID NO: 48] BBRRAA-NH2
75 81.7 5 5.4 [SEQ ID NO: 49] AABBRR-NH2 20-35 21.8-38.1 5 5.4 [SEQ
ID NO: 50] BBAARR-NH2 >150 163.4 5 5.4 [SEQ ID NO: 51]
AAWBRR-NH2 150 170.2 20-35 22.7-39.7 [SEQ ID NO: 52] WBAARR-NH2
>300 340.5 35 39.7 [SEQ ID NO: 53] WBRRAA-NH2 >300 340.5 75
85.1 [SEQ ID NO: 54] AWRBRA-NH2 Pmc+ 10 8.7 2.5 2.2 [SEQ ID NO: 55]
AAWRBR-NH2 Pmc+ 15 13.1 2.5 2.2 [SEQ ID NO: 56] RRAAWW-NH2 Pmc+ 20
17.4 2.5 2.2 [SEQ ID NO: 57] WRBRAA-NH2 Pmc+ 15 13.1 5 4.4 [SEQ ID
NO: 58] RRAAWB-NH2 150-300 170.2-340.5 10 11.3 [SEQ ID NO: 59]
AWRBRA-NH2 35 39.7 35 39.7 [SEQ ID NO: 60] WRAABR-NH2 150 170.2 35
39.7 [SEQ ID NO: 61] WRBRAA-NH2 150 170.2 35 39.7 [SEQ ID NO: 62]
AAWRBR-NH2 150-300 170.2-340.5 35 39.7 [SEQ ID NO: 63] B =
biphenylalanine
[0276] This data shows excellent activity, particularly for those
peptides having the super bulky Pmc group against the Gram positive
bacteria. The data also shows the actual sequence is not highly
significant. Surprisingly and advantageously, the smaller peptides
are more active c.f. WBRR [SEQ ID NO:48] and WBRRAA [SEQ ID
NO:54].
Example 6
[0277] Described below are general procedures for peptide coupling,
deprotection and purification as used, or suitable for use, in
preparing the peptides described herein.
[0278] Peptide Coupling General Procedure
[0279] Synthesis
[0280] The N-Boc protected amino acid derivative (1.05 eq) and
C-terminal protected (either as methyl ester, benzyl ester,
biphenylmethyl ester or beta-naphtyl amide) amino acid derivative
(1.00 eq) and 1-hydroxybenzotriazole (HOBT) (1.2 eq) were added to
the reaction vessel. Diisopropylethylamine (DIPEA) (2.4 eq) and
dimethyl-formamide (DMF) (5 ml/mmol N-Boc protected amino acid) was
added. The reaction mixture was stirred until all components were
dissolved. O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HBTU) (1.2 eq) was added portionwise. The
reaction mixture was shaken for 1 h.
[0281] Extraction and Work-Up
[0282] The reaction mixture from a 1 mmol batch was diluted with
ethyl acetate (16 ml) and washed twice with a mixture of 12 ml 5%
citric acid and 5 ml brine. The subsequent organic phase was washed
twice with a mixture of 6 ml saturated sodium bicarbonate and 2 ml
brine.
[0283] Cleavage of the N-Boc Protected Peptide
[0284] A suitable procedure is described earlier in these
Examples.
[0285] Purification and Analysis of the Peptides
[0286] The peptides were purified on an RP-HPLC C18-column
(Delta-Pak C18, 100 .ANG., 15 .mu.m, 25.times.100 mm, Waters
Corporation, Milford, Mass., USA) using a mixture of water and
acetonitrile (containing 0.1% TFA) as mobile phase and employing
UV-detection at 254 nm. All peptides were analyzed for impurities
on an analytical RP-HPLC C18-column (Delta-Pak C18, 100 .ANG., 5
.mu.m, 3.9.times.150 mm, Waters Corporation) with a mixture of
water and acetonitrile (containing 0.1% TFA) as mobile phase.
Purity of all peptides was found to be above 96%. The integrity of
the peptides was checked by positive ion electrospray ionization
mass spectrometry on a VG Quattro quadrupole mass spectrometer (VG
Instruments Inc., UK).
Example 7
[0287] Preparation of H-Arg-Obzl (after Bodanszky, M and Bodanszky,
A, "The practice of peptide synthesis" (1994) Springer Verlag, p.
30-31)
[0288] Water (2 ml) was added to a solution of Boc-Arg-OH (2.5
mmol) in methanol (20 ml). The solution was neutralised with a 20%
solution of Cs.sub.2CO.sub.3 in water and then evaporated in vacuo
to dryness. Residual water was removed by repeated addition and
evaporation of toluene. The solid cesium salt of Boc-arginine was
treated with DMF (25 ml) and benzyl bromide (3 mmol) and stirred at
room temperature for 6 h. The DMF was removed in vacuo and the
product was dissolved in acetone and filtered. The filtrate was
evaporated in vacuo and the product was treated with 95%
trifluoroactetic acid (TFA) (4 ml). The resulting product
H-Arg-OBzl was isolated by tituration by diethyl ether. The salt of
H-Arg-OBzl was isolated by treating the product with
para-toluenesulfonic acid (5 mmol) in ether.
[0289] Preparation of H-Arg-OBip (after Bodanszky, M and Bodanszky,
A, "The practice of peptide synthesis" (1994) Springer Verlag, p.
30-31)
[0290] Water (2 ml) was added to a solution of Boc-Arg-OH (2.5
mmol) in methanol (20 ml). The solution was neutralised with a 20%
solution of Cs.sub.2CO.sub.3 in water and then evaporated in vacuo
to dryness. Residual water was removed by repeated addition and
evaporation of toluene. The solid cesium salt of Boc-arginine was
treated with DMF (25 ml), biphenylmethylchloride (3 mmol) and
potassium iodide (1 mmol) and stirred at room temperature for 6 h.
The DMF was removed in vacuo and the product was dissolved in
acetone and filtered. The filtrate was evaporated in vacuo and the
product was treated with 95% trifluoroactetic acid (TFA) (4 ml).
The resulting product H-Arg-OBip was isolated by tituration by
diethyl ether. The salt of H-Arg-OBip was isolated by treating the
product with para-toluenesulfonic acid (5 mmol) in ether.
Example 8
[0291] The following C terminally modified dipeptides were also
made and tested. For convenience, their chemical structures are
given below
##STR00006## ##STR00007##
TABLE-US-00010 TABLE 9 Minimum inhibitory concentrations in
.mu.g/ml Peptide E. coli S. aureus P. aeruginosa MRSA MRSE AROBip
300 100 37.5 25 FROBip 100 12.5 12.5 5 BipROBip 50 5.0 5.0 5.0
FR.beta.NA >150 5 >300 12.5 5 RW.beta.NA 100 12.5 12.5 12.5
.beta.NA = beta-naphtylamine
Example 9
[0292] Peptidomimetics based on KWOBzl have been prepared and
tested to demonstrate that a peptide structure is not required for
activity, provided the desired structural motifs are present.
##STR00008##
[0293] The first compound was obtained from Neosystems in France.
The second compound was prepared from indoleacetic acid using
standard techniques (Cs salt mediated esterification of the acid
with benzyl bromide) ane coupling to diBoc lysine using a standard
coupling protocol.
TABLE-US-00011 TABLE 10 Minimum inhibitory concentrations in
.mu.g/ml P. S. Peptide E. coli aeruginosa aureus MRSA MRSE KW-OBzl
>300 37.5 37.5 25 kW-OBzl >300 100 K.psi.(CH.sub.2NH)WOBzl
>300 300 K.psi.(COO)WOBzl 150 300 50 100 37.5 K.psi.(COO)wOBzl
150 300 50 100 37.5 Lower case letters denote D-enantiomers
[0294] These results indicate that the ester derivatives are at
least as active as their peptide equivalents and illustrates the
benefits of a carbonyl group.
Example 10
[0295] The following diphenylethylene diamines available from the
Aldrich catalogue were also tested and all had a MIC value against
S. aureus of 250 .mu.g/ml.
##STR00009##
Example 11
[0296] The following molecules consisting of a modified single
super bulky amino acid were made and tested for their antimicrobial
activity.
##STR00010##
[0297] Boc-Tbt-Dab-Z:
[0298] A mixture of Boc-Tbt-OH (0.7510 g, 1.6 mmol),
N--Z-1,4-diaminobutane mono-hydrochloride (0.4323 g, 1.7 mmol),
HOBt (0.8715 g, 5.7 mmol), DIPEA (1.63 ml, 9.5 mmol) in 12 ml
DMF/CH.sub.2Cl.sub.2 (1:1) is stirred in an ice/water bath and HBTU
(0.7220 g, 1.9 mmol) is added in small portions over 10 min. The
mixture is stirred for another 30 min, the cooling bath is removed
and stirring is continued for 2 hrs and 30 min. The reaction
mixture is evaporated in vacuo. The resulting liquid oil is
dissolved in dichloro-methane and subsequently extracted 3.times.5
ml saturated NaHCO.sub.3, 2.times.5 ml 10% citric acid, 10 ml water
and 5 ml saturated NaCl, before it was dried over MgSO.sub.4 and
evaporated to an light yellow oil. The oil is triturated with water
and dried in vacuo. Purification of the crude product by flash
chromatography (6% MeOH--CHCl.sub.3) afforded 0.96 g (89%) of the
title compound.
[0299] Boc-Tbt-Dae-Z and Boc-Tbt-Dah-Z were prepared by the same
procedure as described for Boc-Tbt-Dab-Z. Crude products were
obtained in almost quantitative yield, and purification by flash
chromatography was not necessary.
[0300] Removal of the Z-protecting group was performed by over
night hydrogenation (1 atm) over 10% Pd on charcoal in
methanol/water (19:1). The catalyst was removed by filtration
through Celite. Evaporation of the solvent in vacuo afforded the
free amine as an yellow oil. The Boc-protecting group was removed
by treatment with Reagent K. The deprotected Boc-diamine was
isolated as a white solid by treating the oily residue after
evaporation of the reaction mixture in vacuo with p-toluensulfonic
acid in diethyl ether. The crude products were purified by RP-HPLC,
and lyophilized to white powders.
[0301] Abbreviations:
[0302] Tbt: .beta.-(2,5,7-tri-tert-butylindol-3-yl)alanine
[0303] Dae: 1,2-diaminoethane
[0304] Dab: 1,4-diaminobutane
[0305] Dah: 1,6-diaminohexane
TABLE-US-00012 TABLE 11 Antimicrobial activity (as MIC in .mu.g/ml)
of Tbt-diamine amides Compound MIC E. coli MIC S. aureus
H-Tbt-Dae-H 15 15 H-Tbt-Dab-H 35 35 H-Tbt-Dah-H 15 15
Example 12
[0306] Several original structures, embodying the combination of a
bulky lipophilic group and a polar residue, can be easily accessed
from cheap raw material such as cyclopentadiene. Two such compounds
are shown below already demonstrating the versatility of the
cyclopentane-based scaffold. Indeed, a simple change in the order
of addition of the bulky or the polar groups leads to two different
products 1 and 2.
##STR00011##
[0307] The synthetic route followed for the preparation of compound
1, and applicable to the preparation of 2, is depicted below.
##STR00012##
[0308] Cyclopentadiene 3 was reacted with singlet oxygen, generated
in situ by photolysis of oxygen in the presence of rose bengale as
the photosensitiser, affording the corresponding endoperoxide. This
peroxide was not isolated but reduced directly by the thiourea
present in the reaction mixture, into the desired cis-diol 4 in an
overall yield of 48%. Esterification of the cis-diol 4 using an
excess of bromoacetyl bromide in the presence of pyridine and DMAP
afforded the desired diester 5 in 40% yield. The subsequent
transformation of diester 5 into the advanced intermediate 6 was
smoothly accomplished by nucleophilic substitution using the
potassium anion of phthalimide. Alkene 6 was then dihydroxylated
from the .alpha.-face under classical osmium-catalysed conditions,
leading to the desired diol 7 in essentially quantitative yield.
The conversion of diol 7 into the final product 1 was effected by
DCC-mediated coupling of 7 with indole carboxylic acid followed by
deprotection of the phthaimido protecting group by hydrazine
hydrate in hot ethanol. The inverse sequence was followed to
prepare 2.
[0309] This versatile sequence can be transposed to the preparation
of a variety of analogues by modifying the order of the addition of
the bulky and polar functions, by altering the relative
stereochemistry of the four hydroxyl functions substituting the
cyclopentane skeleton and by changing the size and nature of the
bulky and polar groups. This strategy is illustrated by some
structures shown below but this is by no means an exhaustive
list.
##STR00013##
Experimental Procedures
1,3-dihydroxy-4-cyclopentene preparation
TABLE-US-00013 ##STR00014## [0310] Reagents ##STR00015## MeOH
Thiourea Rose Bengal ##STR00016## m.w. 66 32 76.11 1017.8 100
Purity 0.802 0.791 d/C Aspect liq. liq. white solid red solid
Equiv. 1 0.68 0.002 Weight 6.416 g 5.03 g 197 mg 9.72 g Moles 97.2
mmoles 66.1 mmoles 0.194 mmoles Vol. 8 ml 1.81 Formula
C.sub.5H.sub.6 CH.sub.3OH CSN.sub.2H.sub.4
C.sub.20H.sub.2Cl.sub.4I.sub.4Na.sub.2O.sub.5 b.p. 43.degree. C.
65.degree. C.
[0311] To a solution of thiourea (5.03 g; 0.68 eq.) and Rose Bengal
(197 mg; 0.002 eq.) in distilled methanol (1.81) was added 8 ml of
freshly distilled cyclopentadiene (1 eq.) at 0.degree. C. Oxygen
was passed through the solution. After 2 h, it was irradiated with
a 450 W mercury lamp and the flux of oxygen was maintained over 2
h. Then, the solution was kept in the dark and the oxygen was
turned off overnight. The mixture was concentrated to 200 ml and
filtrated through charcoal and Celite.RTM. several times until it
was colorless. Then, it was dried on Na.sub.2SO.sub.4 and the
solvent was removed under reduce pressure without heating. The
crude product was purified by horizontal distillation (115.degree.
C., 10.sup.-4 mbar) to obtain 3,525 g (36% yield) of a yellow low
melting point solid.
[0312] NMR H.sup.1 DMSO 300 MHz .delta. in ppm (multiplicity): 1.53
(dt); 2.82 (dt); 4.67 (d); 5.16 (s); 6.03 (s)
[0313] Dibromo-Diester Preparation
TABLE-US-00014 ##STR00017## Reagents ##STR00018## Bromoacetic
bromide Pyridine DMAP DCM Product m.w. 100 202 79 122.12 341 Purity
d/C 2.317 0.978 1.325 Aspect Yellow liq. liq. white liq. solid
Equiv. 1 3 3 0.08 Weight 7.05 g 42.622 g 16.67 g 0.7 g Moles 70.5
mmoles 211 mmoles 211 mmoles 5.7 mmoles 24.04 g Vol. 18.39 ml 17.04
ml 106 ml Formula C.sub.5H.sub.8O.sub.2 C.sub.2H.sub.2Br.sub.2O
C.sub.5H.sub.6N C.sub.7H.sub.10N.sub.2 CH.sub.2Cl.sub.2 b.p.
115.degree. C. 147.degree. C. 115.degree. C. 40.degree. C.
(10.sup.-4 mbar)
[0314] To a solution of 7.05 g (1 eq.) of diol and 46.62 g (3 eq.)
of bromoacetic bromide in 250 ml of dichloromethane was added 17.04
ml (3 eq.) of pyridine and 700 mg (0.08 eq.) of DMAP at 0.degree.
C. The solution was allowed to warm to room temperature and was
maintained under agitation overnight. Then, 700 ml of DCM were
added and the organic phase was washed with 70 ml of 3M HCl, 140 ml
of a saturated solution of Na.sub.2CO.sub.4, and 140 ml of water.
The organic layer was dried on Na.sub.2SO.sub.4, filtrated and the
solvent was remove under reduce pressure. The crude product was
purified by a Flash chromatography (AcOEt/EP 35:75) to obtain 9.76
g (40.6% yield) of a colorless liquid.
[0315] NMR H.sup.1 CDCl.sub.3 300 MHz .delta. in ppm
(multiplicity): 1.8 (dt); 2.89 (dt); 3.91 (s); 5.58 (dd); 6.13
(s)
[0316] Diphtalimido-Diester Preparation
TABLE-US-00015 ##STR00019## Reagents Dibromo Potassium compound
phtalamide DMSO Diphtalimido m.w. 341 185.22 473 Purity d/C Aspect
liq. white solid Equiv. 1 2.5 Weight 1.311 g 1.78 g 1.816 Moles
3.84 mmoles 9.61 mmoles Vol. 30 ml Formula
C.sub.9H.sub.10Br.sub.2O.sub.4 C.sub.8H.sub.4KNO.sub.2
C.sub.2H.sub.6SO b.p. 189.degree. C.
[0317] A solution of 1.311 g (1 eq.) of the dibromo compound and
1.78 g (2.5 eq.) of potassium phtalamide in 30 ml of DMSO was kept
under reflux overnight. The mixture was diluted with 250 ml of
diethylether and was washed 3 times by 150 ml of brine. The organic
layer was dried on Na.sub.2SO.sub.4, filtrated and the solvent was
removed under reduce pressure. The crude product was purified by a
Flash chromatography (AcOEt/EP 80:20) to obtain 1.24 g (68.8%
yield) of a white solid.
[0318] NMR H.sup.1 CDCl.sub.3 300 MHz .delta. in ppm
(multiplicity): 1.85 (dt); 2.87 (dt); 4.42 (s); 5.61 (dd); 6.11
(s); 7.85 (m)
Sequence CWU 1
1
6316PRTArtificialmembrane acting anti-microbial agent molecule 1Trp
Arg Trp Arg Trp Arg1 526PRTArtificialmembrane acting anti-microbial
agent molecule 2Arg Arg Arg Trp Trp Trp1 536PRTArtificialmembrane
acting anti-microbial agent molecule 3Arg Trp Trp Trp Arg Arg1
546PRTArtificialmembrane acting anti-microbial agent molecule 4Trp
Trp Arg Arg Arg Trp1 556PRTArtificialmembrane acting anti-microbial
agent molecule 5Arg Trp Arg Trp Arg Trp1 566PRTArtificialmembrane
acting anti-microbial agent molecule 6Arg Trp Arg Tyr Arg Trp1
575PRTArtificialmembrane acting anti-microbial agent molecule 7Trp
Arg Trp Arg Trp1 585PRTArtificialmembrane acting anti-microbial
agent molecule 8Trp Arg Tyr Arg Trp1 595PRTArtificialmembrane
acting anti-microbial agent molecule 9Arg Trp Arg Trp Arg1
5105PRTArtificialmembrane acting anti-microbial agent molecule
10Trp Arg Trp Arg Tyr1 5114PRTArtificialmembrane acting
anti-microbial agent molecule 11Arg Trp Trp
Arg1124PRTArtificialmembrane acting anti-microbial agent molecule
12Trp Arg Arg Trp1134PRTArtificialmembrane acting anti-microbial
agent molecule 13Trp Arg Trp Arg1143PRTArtificialmembrane acting
anti-microbial agent molecule 14Trp Arg
Trp1153PRTArtificialmembrane acting anti-microbial agent molecule
15Arg Trp Arg1164PRTArtificialmembrane acting anti-microbial agent
molecule 16Arg Trp Arg Trp1174PRTArtificialmembrane acting
anti-microbial agent molecule 17Arg Trp Arg
Trp1184PRTArtificialmembrane acting anti-microbial agent molecule
18Arg Trp Xaa Xaa1194PRTArtificialmembrane acting anti-microbial
agent molecule 19Arg Trp Xaa Xaa1204PRTArtificialmembrane acting
anti-microbial agent molecule 20Arg Trp Trp
Arg1214PRTArtificialmembrane acting anti-microbial agent molecule
21Arg Trp Trp Arg1222PRTArtificialmembrane acting anti-microbial
agent molecule 22Arg Trp1232PRTArtificialmembrane acting
anti-microbial agent molecule 23Xaa Xaa1242PRTArtificialmembrane
acting anti-microbial agent molecule 24Trp
Arg1253PRTArtificialmembrane acting anti-microbial agent molecule
25Trp Arg Trp1263PRTArtificialmembrane acting anti-microbial agent
molecule 26Xaa Xaa Xaa1273PRTArtificialmembrane acting
anti-microbial agent molecule 27Xaa Arg
Trp1283PRTArtificialmembrane acting anti-microbial agent molecule
28Trp Trp Arg1293PRTArtificialmembrane acting anti-microbial agent
molecule 29Arg Trp Arg1304PRTArtificialmembrane acting
anti-microbial agent molecule 30Arg Trp Arg
Trp1314PRTArtificialmembrane acting anti-microbial agent molecule
31Arg Trp Xaa Xaa1324PRTArtificialmembrane acting anti-microbial
agent molecule 32Arg Trp Trp Arg1334PRTArtificialmembrane acting
anti-microbial agent molecule 33Arg Trp Arg
Trp1344PRTArtificialmembrane acting anti-microbial agent molecule
34Arg Trp Xaa Xaa1356PRTArtificialmembrane acting anti-microbial
agent molecule 35Arg Xaa Arg Tyr Arg Xaa1 5366PRTArtificialmembrane
acting anti-microbial agent molecule 36Ala Ala Trp Trp Arg Arg1
5376PRTArtificialmembrane acting anti-microbial agent molecule
37Arg Arg Ala Ala Trp Trp1 5386PRTArtificialmembrane acting
anti-microbial agent molecule 38Ala Trp Arg Trp Arg Ala1
5396PRTArtificialmembrane acting anti-microbial agent molecule
39Trp Arg Ala Ala Trp Arg1 5406PRTArtificialmembrane acting
anti-microbial agent molecule 40Trp Trp Ala Ala Arg Arg1
5416PRTArtificialmembrane acting anti-microbial agent molecule
41Trp Trp Ala Ala Arg Arg1 5426PRTArtificialmembrane acting
anti-microbial agent molecule 42Ala Ala Trp Trp Arg Arg1
5436PRTArtificialmembrane acting anti-microbial agent molecule
43Arg Arg Ala Ala Trp Trp1 5446PRTArtificialmembrane acting
anti-microbial agent molecule 44Trp Arg Ala Ala Trp Arg1
5456PRTArtificialmembrane acting anti-microbial agent molecule
45Ala Trp Arg Trp Arg Ala1 5464PRTArtificialmembrane acting
anti-microbial agent molecule 46Xaa Xaa Arg
Arg1474PRTArtificialmembrane acting anti-microbial agent molecule
47Trp Xaa Arg Arg1484PRTArtificialmembrane acting anti-microbial
agent molecule 48Trp Xaa Arg Arg1496PRTArtificialmembrane acting
anti-microbial agent molecule 49Xaa Xaa Arg Arg Ala Ala1
5506PRTArtificialmembrane acting anti-microbial agent molecule
50Ala Ala Xaa Xaa Arg Arg1 5516PRTArtificialmembrane acting
anti-microbial agent molecule 51Xaa Xaa Ala Ala Arg Arg1
5526PRTArtificialmembrane acting anti-microbial agent molecule
52Ala Ala Trp Xaa Arg Arg1 5536PRTArtificialmembrane acting
anti-microbial agent molecule 53Trp Xaa Ala Ala Arg Arg1
5546PRTArtificialmembrane acting anti-microbial agent molecule
54Trp Xaa Arg Arg Ala Ala1 5556PRTArtificialmembrane acting
anti-microbial agent molecule 55Ala Trp Arg Xaa Arg Ala1
5566PRTArtificialmembrane acting anti-microbial agent molecule
56Ala Ala Trp Arg Xaa Arg1 5576PRTArtificialmembrane acting
anti-microbial agent molecule 57Arg Arg Ala Ala Trp Trp1
5586PRTArtificialmembrane acting anti-microbial agent molecule
58Trp Arg Xaa Arg Ala Ala1 5596PRTArtificialmembrane acting
anti-microbial agent molecule 59Arg Arg Ala Ala Trp Xaa1
5606PRTArtificialmembrane acting anti-microbial agent molecule
60Ala Trp Arg Xaa Arg Ala1 5616PRTArtificialmembrane acting
anti-microbial agent molecule 61Trp Arg Ala Ala Xaa Arg1
5626PRTArtificialmembrane acting anti-microbial agent molecule
62Trp Arg Xaa Arg Ala Ala1 5636PRTArtificialmembrane acting
anti-microbial agent molecule 63Ala Ala Trp Arg Xaa Arg1 5
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